Conjugated antibodies

Abstract

The present invention includes compounds for the prevention and treatment of cancer and ocular angiogenesis having a first binding peptide having anti-angiogenic or anti- cancer activity located at an N-terminus of a heavy chain and / or a light chain polypeptide, wherein the heavy chain polypeptide comprises a heavy chain region of an antibody used to treat cancer or ocular angiogenesis operatively linked to a first immunoglobulin (IgG) antibody peptide and the light chain polypeptide comprises a light chain region of an antibody used to treat cancer or ocular angiogenesis operatively linked to a second IgG antibody peptide.

Description

CONJUGATED ANTIBODIESFIELD OF THE INVENTION

[0001] The invention relates to compounds for the prevention and treatment of cancer and eye diseases caused by ocular angiogenesis.CROSS-REFERENCE TO RELATED APPLICATION

[0002] This application claims priority to US provisional application no. 63 / 779619 filed March 28, 2025, the contents of which are incorporated herein by reference.CROSS-REFERENCE TO RELATED DOCUMENTS

[0003] This application is filed concurrently with a sequence listing named SEQUENCE LISTING 92834968 encoded to meet the WIPO standards.BACKGROUND

[0004] Angiogenesis is a process wherein new blood vessels are grown to deliver more nutrients. This natural process can be co-opted in many diseases including solid tumor cancers to feed the growth of a tumor. Pathological angiogenesis also occurs in eye diseases where ocular angiogenesis can lead to vision loss. As a result, angiogenesis is an important target for treatment of solid tumors and treatment of eye diseases caused by ocular angiogenesis.

[0005] Cancer is a leading cause of death worldwide, second only to cardiovascular disease. For the most part, while leukemias (blood bom cancers) and melanoma (skin cancers) are well served by current treatments, solid tumors have been less well served. As such, the rate of death from solid tumors are a major concern for oncologists and represent a major unmet medical need. Angiogenesis and metastasis are two fundamental processes that are important with respect to solid tumors.

[0006] Cancer metastasis is the major cause of cancer morbidity and mortality and may account for up to 90% of cancer deaths. Although cancer survival rate has been significantly improved over the years, the improvement is primarily due to early diagnosis and cancer growth inhibition. Limited progress has been made in the treatment of cancer metastasis. Current treatments for cancer metastasis are mainly chemotherapy andradiotherapy, though the new generation anti-cancer drugs (predominantly neutralizing antibodies for growth factors and small molecule kinase inhibitors) do have indirect effects on cancer metastasis in addition to their direct effects on cancer growth.

[0007] Ocular angiogenesis is the formation of new blood vessels in the eye that occurs in various eye diseases and can lead to vision loss. Ocular angiogenesis can occur in response to pathological hypoxia in many eye diseases including neovascular age-related macular degeneration (NAMD). Pathological neovascularization in the normally avascular and clear cornea can also result in vision loss.

[0008] There are limited treatment options comprising fusion proteins and uses thereof that limit angiogenesis and / or tumor metastasis. The present invention includes synergistic combinations of peptides and fragments of therapeutic antibodies with anticancer and / or anti-angiogenic activity. These compounds can improve the treatment of cancer and ocular angiogenesis.SUMMARY

[0009] The present invention includes compounds for the treatment of cancer and treatment of ocular angiogenesis.

[0010] In an embodiment of the present invention there is provided a compound comprising a first binding peptide having anti-angiogenic or anti-cancer activity operatively linked to an N-terminus of a heavy chain and / or a light chain polypeptide, wherein the heavy chain polypeptide comprises a heavy chain region of an antibody used to treat cancer or ocular angiogenesis operatively linked to a first immunoglobulin (IgG) antibody peptide and the light chain polypeptide comprises a light chain region of an antibody used to treat cancer or ocular angiogenesis operatively linked to a second immunoglobulin (IgG) antibody peptide.

[0011] The second IgG antibody peptide may be a human kappa constant light (CL) chain.

[0012] The compound of the present invention may comprise two heavy chain polypeptides and two light chain polypeptides, two heavy chain polypeptides and one light chain polypeptide or two heavy chain polypeptides.

[0013] The first IgG antibody peptide may be a fragment of an Fc fusion peptide, theFc fusion peptide may be a human Fc fusion peptide or an IgG protein. The Fc fusion peptide may be selected from the group consisting of an IgG 1 isotype Fc peptide, an lgG2 isotype Fc peptide, an lgG3 isotype Fc peptide, an lgG4 isotype Fc peptide.

[0014] The first IgG antibody peptide may also be a constant heavy chain peptide of human lgG1. The heavy chain peptide of human IgG 1 may have one or more amino acid mutations.

[0015] The first binding peptide of the present invention may be C20. The first binding peptide may be one of L17H, P144 or G3-A3.

[0016] Another embodiment of the present invention further comprises a second binding peptide having anti-angiogenic or anti-cancer activity operatively linked to the N-terminus or C-terminus of the heavy chain and / or the light chain polypeptide. The second binding peptide may be one of C20, L17H, P144 or G3-A3.

[0017] The heavy chain region of an antibody used to treat cancer or ocular angiogenesis may comprise one of Praluent heavy chain, Kevzara heavy chain, Herceptin heavy chain or Avastin heavy chain.

[0018] The light chain region of an antibody used to treat cancer or ocular angiogenesis may comprise one of Praluent light chain, Kevzara light chain, Herceptin light chain or Avastin light chain.

[0019] The present invention may further comprise a third binding peptide comprising an endostatin derived peptide sequence having endostatin activity or a plasminogen derived peptide sequence having plasminogen activity or a hepatocyte growth factor derived peptide having hepatocyte growth factor antagonist activity or a vasostatin derived peptide having vasostatin activity, operatively linked to the C-terminus of the heavy chain polypeptide.

[0020] The third binding peptide may be selected from the group consisting of a fragment of an endostatin having endostatin activity, an endostatin, a fragment of a plasminogen having plasminogen activity, an endostatin with a P125 mutation, a kringle domain of plasminogen, kringle domains 1 to 5 of plasminogen, kringle domain 5 from a plasminogen, NK1 domain of hepatocyte growth factor with a Y124A mutation or vasostatin 1.

[0021] An embodiment of the present invention may have two heavy chain polypeptidescoded by a heavy chain sequence and two light chain polypeptides coded by a light chain sequence selected from a group of heavy chain sequence and light chain sequence combinations comprising SEQ ID No. 1 and SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, SEQ ID No. 1 and SEQ ID. No. 4, SEQ ID No. 5 and SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, SEQ ID No. 12 and SEQ ID No. 10, SEQ ID No. 13 and SEQ ID No.10, SEQ ID No. 14 and SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No. 15, SEQ ID No.17 and SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20, SEQ ID No. 21 and SEQ ID No. 8, SEQ ID No. 22 and SEQ ID No. 10, SEQ ID No. 25 and SEQ ID No. 10, SEQ ID No. 26 and SEQ ID No. 10, SEQ ID No. 27 and SEQ ID No. 10, SEQ ID No. 28 and SEQ ID No. 10, SEQ ID No. 29 and SEQ ID No. 30, SEQ ID No. 9 and SEQ ID No. 30, SEQ ID No. 33 and SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 36, SEQ ID No. 37 and SEQ ID No. 34, SEQ ID No. 38 and SEQ ID No. 39, SEQ ID No. 40 and SEQ ID No. 41, SEQ ID No. 42 and SEQ ID No. 43, SEQ ID No. 44 and SEQ ID No. 43, or SEQ ID No.45 and SEQ ID No. 46.

[0022] A compound may have polypeptide sequences at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the polypeptide sequences of the compound of the present invention.

[0023] The compound of the present invention may be selected from the group consisting of C20H-Herceptin-K5, C20L-Herceptin-K5, C20HL-Herceptin-K5, Tetravalent C20-Fc4, C20H-Kevzara-K5, Praluent-Endo(P125A), C20-Praluent-Endo(P125A), C20-Praluent, L17H-C20L-Avastin-Endo, L17H-C20L-Avastin-K5, L17H-C20L-Kevzara-K5, C20L-Kevzara-L17-Endostatin(P125A), C20H-Kevzara-HGF(Y124Y)NK1 , C20-Praulent-Vasostatinl, C20-Kevzara, C20-Kevzara-K5-L17, C20-Kevzara-Vasostatin1-L17, C20-Kevzara-Endostatin(p125A)-L17, C20L-Praluent-Vasostatin1 , C20L-Praluent, L17H-C20L-Herceptin-K5, (C20-L17)H-Herceptin-K5, C20L-Herceptin-L17, C20L-Kevzara-L17, L17H-C20L-Avastin-L2-P144, L17H-C20L-Kevzara-L1-P144, L17H-C20L-Kevzara-K5-L1-P144, and L17H-C20L-Herceptin-K5-L1-P144.

[0024] The compound of the present invention may comprise Praluent-Endo(P125A). The compound of the present invention may have two heavy chain polypeptides and two light chain polypeptides, wherein the heavy chain polypeptides are coded by a heavy chainsequence comprising SEQ ID. NO. 11 and the light chain polypeptides are coded by a light chain sequence comprising SEQ ID. No. 10.

[0025] The compound of the present invention may be selected from the group consisting of ATF-Fc4-Endostatin(P125A)-L17H, ATF-Fc4-Vasostatin1 , ATF-Arrestin-FC (lgG4) and ATF-hulgG4 Fc-Arrestin.

[0026] The compound of the present invention may comprise C20 Bivalent.

[0027] The compound of the present invention by be selected from the group consisting of C20CD16IL15, C20CD16IL15K5, C20-CD16-1115-Fc1 and C20-CD16-II15-Fc1-K5.

[0028] The compound of the present invention may comprise two heavy chain polypeptides, wherein the heavy chain polypeptides are coded by a heavy chain sequence selected from SEQ ID No. 23, SEQ ID No. 31 , SEQ ID No. 32, SEQ ID No. 51 , SEQ ID No. 52, SEQ ID No. 53 or SEQ ID No. 54.

[0029] The compound of the present invention may comprise a first heavy chain polypeptide coded by a first heavy chain sequence and a second heavy chain polypeptide coded by a second heavy chain sequence selected from a group of first heavy chain sequence and second heavy chain sequence combinations comprising SEQ ID No. 47 and SEQ ID No. 48 or SEQ ID No. 49 and SEQ ID No. 50.

[0030] Also taught are compounds substantially homologous to the above mentioned compounds.

[0031] Also taught are nucleotides encoding the compounds. Also taught are vectors containing the compounds.

[0032] The compounds may exhibit anti-cancer activity. Thus, also taught are methods of treating cancer comprising administration of a composition comprising the compound of the invention, and the use of the compounds for treatment of cancer. The cancer may be a solid cancer. The methods may inhibit metastasis. The method or use may further comprise one or more of the following a) administering chemotherapy; b) administering radiation therapy; or c) administering one or more additional therapeutic agents.

[0033] The compounds may exhibit anti-angiogenic activity to treat eye diseases caused by ocular angiogenesis. Thus, also taught are methods of treating eye diseasescaused by ocular angiogenesis comprising administration of a composition comprising the compound of the invention, and the use of the compounds for treatment of eye diseases caused by ocular angiogenesis.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Reference will now be made, by way of example, to the accompanying drawings which show example embodiments, and in which:

[0035] Figure 1 A provides a general schematic of the constructs which can include peptide ligands (Z and Y) operatively linked to the N-terminus of a heavy (HC) and / or a light chain (LC) of an anti-cancer or anti-angiogenic antibody and further anti-angiogenic binding peptides operatively linked to the C-terminal of the heavy chain of the anti-cancer or anti-angiogenic antibody (X).

[0036] Figure 1B provides an example of a construct where a C20 ligand is bound to the N-terminal of an anti-cancer or anti-angiogenic antibody heavy chain (HC) and K5 peptide bound to the C-terminal of anti-cancer or anti-angiogenic antibody HC.

[0037] Figure 2 shows an SDS-PAGE analysis of C20H-Herceptin-K5.

[0038] Figure 3 shows an SDS-PAGE analysis of C20L-Herceptin-K5.

[0039] Figure 4 shows an SDS-PAGE analysis of C20HL-Herceptin-K5.

[0040] Figure 5 shows an SDS-PAGE analysis of Tetravalent C20-Fc4.

[0041] Figure 6 shows an SDS-PAGE analysis of C20H-Kevzara-K5.

[0042] Figure 7 shows an SDS-PAGE analysis of Praluent Control.

[0043] Figure 8 shows an SDS-PAGE analysis of Praluent-Endo(P125A).

[0044] Figure 9 shows an SDS-PAGE analysis of C20-Praluent-Endo(P125A).

[0045] Figure 10 shows an SDS-PAGE analysis of C20-Praluent.

[0046] Figure 11 is a graph showing normalized tumor size as effected by various ATF compounds.

[0047] Figure 12 is a graph showing normalized tumor size as effected by various C20 and Praluent constructs.

[0048] Figure 13 is a graph showing normalized tumor size as effected by various constructs.

[0049] Figure 14 is a graph showing the number of metastases as effected byvarious constructs.

[0050] Figure 15 is a graph showing normalized tumor size as effected by C20-Herceptin and C20-Kevzara constructs.

[0051] Figure 16 shows an SDS-PAGE analysis of L17H-C20L-Kevzara-K5.

[0052] Figure 17 shows an SDS-PAGE analysis of C20L-Kevzara-L17-Endostatin(P125A).

[0053] Figure 18 shows an SDS-PAGE analysis of ATF-Fc4-Endostatin(P125A)-L17H.

[0054] Figure 19 shows an SDS-PAGE analysis of Kevzara Control.

[0055] Figure 20 shows an SDS-PAGE analysis of C20-Kevzara.

[0056] Figure 21 shows an SDS-PAGE analysis of C20-Kevzara-K5-L17.

[0057] Figure 22 shows an SDS-PAGE analysis of L17H-C20L-Herceptin-K5.

[0058] Figure 23 shows an SDS-PAGE analysis of (C20-L17)H-Herceptin-K5.

[0059] Figure 24 shows an SDS-PAGE analysis of C20L-Herceptin-L17.

[0060] Figure 25 shows an SDS-PAGE analysis of C20L-Kevzara-L17.

[0061] Figure 26 is a graph showing the percentage viability of cancer cell line MDA-MB-231 when treated with various constructs as compared to the negative control.

[0062] Figure 27 shows an SDS-PAGE analysis of L17H-C20L-Avastin-L2-P144.

[0063] Figure 28 shows an SDS-PAGE analysis of C20CD16IL15.

[0064] Figure 29 shows an SDS-PAGE analysis of C20CD16IL15K5.

[0065] Figure 30 shows an SDS-PAGE analysis of C20-CD16-1115-Fc1.

[0066] Figure 31 shows an SDS-PAGE analysis of C20-CD16-1115-Fc1-K5.

[0067] Figure 32 shows an SDS-PAGE analysis of ATF-Arrestin-FC (lgG4).

[0068] Figure 33 shows an SDS-PAGE analysis of ATF-hulgG4 Fc-Arrestin.DETAILED DESCRIPTION

[0069] Throughout the following description specific details are set forth to provide a more thorough understanding to persons skilled in the art. However, well-known elements may not have been shown or described in detail. Accordingly, the description and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[0070] The present invention relates to the combination of short peptide sequencesthat target the tumor microenvironment or have anti-angiogenic activity with existing antibodies that treat cancer or ocular angiogenesis.

[0071] In an embodiment of the present invention there is provided a compound comprising a first binding peptide having anti-angiogenic or anti-cancer activity operatively linked to an N-terminus of a heavy chain and / or a light chain polypeptide, wherein the heavy chain polypeptide comprises a heavy chain region of an antibody used to treat cancer or ocular angiogenesis operatively linked to a first immunoglobulin (IgG) antibody peptide and the light chain polypeptide comprises a light chain region of an antibody used to treat cancer or ocular angiogenesis operatively linked to a second immunoglobulin (IgG) antibody peptide.

[0072] The compound of an embodiment of the present invention may comprise two heavy chains and two light chains, two heavy chains and one light chain or two heavy chains. The second IgG antibody peptide may be a human kappa constant light (CL) chain.

[0073] The first IgG antibody peptide may be a fragment of an Fc fusion peptide, the Fc fusion peptide may be a human Fc fusion peptide or an IgG protein. The Fc fusion peptide may be selected from the group consisting of an IgG 1 isotype Fc peptide, an lgG2 isotype Fc peptide, an lgG3 isotype Fc peptide, an lgG4 isotype Fc peptide.

[0074] The isotype of the Fc domain (i.e. IgG 1 , 2, 3 or 4) can be selected pending a preference of improved half-life and / or ADCC and complement activity is desired. For example, in some situations, an IgG 1 isotype may be preferred to increase the anti-cancer activity of the antibody by increasing ADCC. In other situations, an lgG4 isotype may be preferred for example to reduce ADCC which may allow for less toxicity and thereby permit higher doses. In some cases, an lgG3 Fc which is intermediate in ADCC activity to either IgG 1 or lgG4 may be preferred. Note the Fc domain forms a natural homodimer.

[0075] The first IgG antibody peptide may also be a constant heavy chain peptide of human lgG1. The heavy chain peptide of human IgG 1 may have one or more amino acid mutations.

[0076] The invention teaches nucleotides encoding the compounds and vectors containing the compounds. Accordingly, the present invention also provides DNA, RNA and mRNA based treatments. DNA may be administered, reach the damaged cells, enterthe cell and express the proteins of the invention. Multiple delivery techniques can be used. For example, DNA may be entered into an engineered virus to deliver the DNA into a chromosome. Naked DNA approaches can also be used. Alternatively, one can administer a gene that causes a needed protein to be expressed.

[0077] In somatic cell gene therapy (SCGT), the therapeutic genes are transferred into any cell other than a gamete, germ cell, gametocyte, or undifferentiated stem cell. Therapeutic DNA (either integrated in the genome or as an external episome or plasmid) is used to treat disease.

[0078] In germline gene therapy (GGT), germ cells (sperm or egg cells) are modified by the introduction of functional genes into their genomes. Modifying a germ cell causes all the organism's cells to contain the modified gene. The change is therefore heritable and passed on to later generations.

[0079] In in vivo gene therapy, a vector (typically, a virus) is introduced to the patient, which then achieves the desired biological effect by passing the genetic material (e.g. for the therapeutic protein) into the patient's cells. In ex vivo gene therapies, such as CAR-T therapeutics, the patient's own cells (autologous) or healthy donor cells (allogeneic) are modified outside the body (hence, ex vivo) using a vector to express a particular protein, such as a chimeric antigen receptor.

[0080] One of the major events that underlies metastasis is the proteolytic degradation of the extracellular matrix (ECM) to promote tumor cell invasion, migration, and homing to distant organs. Even though several protease systems are implicated in this process, a large body of evidence identified the urokinase-type plasminogen activator receptor (uPAR) system as a central player in mediating proteolysis during cancer invasion and metastasis. The uPA-mediated degradation of the ECM is also crucial for the initiation of angiogenesis.

[0081] uPAR is a is a GPI-anchored cell membrane receptor that localizes urokinase (uPA) proteolytic activity on the cell surface. Its main function is focusing of urokinase (uPA) proteolytic activity, responsible for degradation of ECM components, on the cell surface. uPAR expression is increased in many human cancers and correlates with a poor prognosis and early invasion and metastasis and has been reported on as many asthe complex, and unoccupied UPAR may be recycled to the surface.

[0082] There are two broad mechanisms to inhibit the function of LIPAR; (1) binding of uPA with uPAR, and (2) preventing the cell signaling that can occur by LIPAR which involves both uPA binding and its subsequent binding with co-receptors such as integrins.

[0083] The binding of uPA with uPAR is instrumental for the activation of plasminogen to plasmin, which in turn initiates a series of proteolytic cascade to degrade the components of the extracellular matrix, and thereby cause tumor cell migration from the primary site of origin to a distant secondary organ.

[0084] uPA is often separated into two regions. The first region which is responsible for binding of uPA to UPAR is the amino terminal fragment (ATF). The ATF can be further divided into two domains: an epidermal growth factor (EGF)-like domain and a kringle domain. The “G” domain is the EGF-like region and the “K” is the kringle domain. The majority of the binding occurs with just the EGF-like domain alone, but the entire ATF region including both the EGF-like domain and the kringle domain does have even greater binding the just the EGF-like domain alone.

[0085] The second region of uPA contains the enzymatic activity of uPA. It is initially an inactive pro-enzyme which when activated, enzymatically cleaves plasminogen and activates that enzyme to plasmin which mediates degradation of the extracellular matrix.

[0086] Once uPA binds to UPAR, the complex of the two proteins is also able to interact with other proteins (co-receptors) at the surface of the cancer cell such as integrins. When uPAR interacts with these coreceptors, mostly through Domain 3 of its three domains, this results in activation of cell signaling pathways that hasten the cancer progression including metastases.

[0087] An ATF-Fc fusion (Chinese patent No. CN101050237) has been shown to reduce tumor growth and metastases (Hu XW, Duan HF, Gao LH, et al. Inhibition of tumor growth and metastasis by ATF-Fc, an engineered antibody targeting urokinase receptor. Cancer Biol Then 2008;7(5):651-659. doi:10.4161 / cbt.7.5.5643). Further, an ATF conjugated to the Fc fragment of human immunoglobulin G1 in combination with trastuzumab, a drug that targets the HER2 oncogene, synergistically inhibited HER2-positive breast cancer growth and metastasis in a mouse xenograft model. (Zhou H, Wang H, Yu G, Wang Z, Zheng X, Duan H and Sun J. Synergistic inhibitory effects of anengineered antibody-like molecule ATF-Fc and trastuzumab on tumor growth and invasion in a human breast cancer xenograft mouse model. Oncology Letters 14:5189-5196).

[0088] An antibody (2G10) was identified from a phage display library that prevented uPA association with uPAR, been reported to prevent uPAR and uPA binding(Duriseti S, Goetz DH, Hostetter DR, LeBeau AM, Wei Y, Craik CS. Antagonistic anti-urokinase plasminogen activator receptor (uPAR) antibodies significantly inhibit uPAR-mediated cellular signaling and migration. J Biol Chem. 2010;285(35):26878-26888. doi:10.1074 / jbc.M109.077677). Non-natural smaller peptides which inhibit the uPA / uPAR interaction have been identified from combinatorial libraries as well.

[0089] The first binding peptide of the present invention may be peptide C20 which binds uPAR and inhibits uPAR activity. Short peptide sequences unrelated to the native ATF sequence have been identified which also compete with uPA binding to uPAR (Goodson RJ, Doyle M V., Kaufman SE, Rosenberg S. High-affinity urokinase receptor antagonists identified with bacteriophage peptide display. Proc Natl Acad Sci U S A.1994;91 (15):7129-7133. doi:10.1073 / pnas.91.15.7129). In Goodson et.al., c20, a small linear peptide sequence, had the strongest binding affinity for uPAR out of a screened 15-mer random peptide library displayed on bacteriophage M13.

[0090] There are reports of combining inhibitors of uPA-uPAR (e.g. ATF) with inhibitors such as protease inhibitor domains as well as the anti-angiogenic functional domains of vasostatin. Similarly, there have been reports of Endostatin (P125A) fused to the C-terminus of other antibodies such as Herceptin. WO2023168531 A1 discloses compounds that combined a uPAR antagonist with additional anti-angiogenic activity conferred by endostatin and / or plasminogen derived sequences in the same Fc fusion protein. These compounds reduced tumor formation and metastatic activity in a zebrafish xenograft model. However, there is a need in the art to combine the anti-angiogenic and anti-metastatic activity of short peptide uPAR antagonists with antibodies used to treat cancer or ocular angiogenesis to create compounds with enhanced anti-cancer or anti-angiogenic activity.

[0091] The first binding peptide of the present invention may be C20. The first binding peptide may be one of L17H, P144 or G3-A3.

[0092] Angiopoietin-2 (Ang2) is a ligand that binds to the Tie2 receptor and stimulates angiogenesis. Ang 2 is elevated at sites of normal and pathological angiogenesis. Ang2 selective inhibitors were designed using phage display peptide libraries and FAB libraries against human Ang2 to create peptide-Fc fusion proteins and antibodies. One peptide, L17, and its peptide-Fc fusion protein were potent neutralizers of the Ang2:Tie receptor interaction. Blocking Ang2 activity with antibodies or peptide-Fc fusion proteins reduced tumor growth in mice through an anti-angiogenic mechanism. The anti-ang2 agents also prevented angiogenesis in a rat corneal model. (Oliner J et.al. Suppression of angiogenesis and tumor growth by selective inhibition of angiopoietin-2. Cancer Cell Volume 6, Issue 5, November 2004, 507-516).

[0093] Transforming growth factor beta 1 (TGF-[31) plays an important role in fibrogenesis. TGF-[31 also participates in cell growth and differentiation, formation of the ECM including the deposition of collagen and immune responses. Synthetic peptide inhibitors of TGF-[31 were screened in a model of rat fibrotic liver and P144 blocked TGF-[31 activity and showed anti-fibrotic activity in rats. (Ezquerro et.al. A synthetic peptide from transforming growth factor [3 type III receptor inhibits liver fibrogenesis in rats with carbon tetrachloride liver injury. Cytokine Volume 22, Issues 1-2, April 2003, 12-20). TGF-[31 plays a role in promoting tumor growth and angiogenesis in cancer. TGF-[31 inhibitors have shown promise in treating cancer. (Kim et.al. Novel therapies emerging in oncology to target the TGF-[3 pathway Journal of Hematology & Oncology volume 14:55 (2021))

[0094] Galectin-3 is a member of the galectin family of soluble animal lectins and plays a role in metastasis through carbohydrate-mediated metastatic adhesion interactions. Peptide antagonists of Galectin-3 have been developed using phage display libraries, including G3-A3. These peptide antagonists inhibit the adhesion of breast cancer cells to endothelial cells in vitro. (Jun Zou, Characterization of peptides and phage that bind galectin-3 selected from bacteriophage display libraries: a study of the role of galectin-3 in metastasis-associated cancer cell adhesion. University of Missouri Dissertation)

[0095] Monoclonal antibody therapeutics with up to five specificities have been engineered by fusing peptides to the N-terminus and C-terminus of the full light and heavy chains of trastuzumab. Trastuzumab antibodies with ErbB2 and Ang2 targeting peptidesshowed enhanced anti-tumor efficacy in a mouse xenograft model that is angiogenesisdependent. However, these antibodies only have specificities for ErbB2, EGFR, IGF-1 R, Ang2 and integrin av[33 and use the entire heavy and light chains of trastuzumab. These engineered antibodies use larger sequences (domains) compared to the present invention. Further, embodiments of the present invention use the entire heavy and light chains of the therapeutic antibody. (LaFluer et.al. Monoclonal antibody therapeutics with up to five specificities: functional enhancement through fusion of target-specific peptides.2013 Mar-Apr;5(2):208-18.)

[0096] The heavy chain region of an antibody used to treat cancer or ocular angiogenesis may comprise one of Praluent heavy chain, Kevzara heavy chain, Herceptin heavy chain or Avastin heavy chain. The light chain region of an antibody used to treat cancer or ocular angiogenesis may comprise one of Praluent light chain, Kevzara light chain, Herceptin light chain or Avastin light chain.

[0097] Praluent is an antibody that inhibits proprotein convertase subtilisin / kexin— 9 (PCSK9). PCSK9 plays a critical role in cholesterol metabolism and therefore Praluent is a promising therapy for cardiovascular diseases to reduce cholesterol. Angiogenic inhibitors such as Endostar, a recombinant form of endostatin, have been shown to inhibit carotid plaque neovascularization in patients with non-small cell lung cancer (NSCLC). (Pu et.al. The Therapeuatic Effect of Endostar on Soft Carotid Plaque Neovascularization in Patients with Non-small Cell Lung Cancer. Sci Rep. 2015 Mar 10;5:8956). Further, uPAR plays a role in atherosclerosis partially through the modulation of monocyte function. (Hindy et.al. Increased soluble urokinase plasminogen activator levels modulate monocyte function to promote atherosclerosis J Clin Invest. 2022 Dec 15; 132(24)). It has been shown that PCSK9 inhibition has anti-cancer effects in colorectal cells. (Oza and Kashfi, The evolving landscape of PCSK9 inhibition in cancer. European Journal of Pharmacology (2023) 949, 175721) However, there is need in the art for a compound that can inhibit PCSK9 and uPAR to improve anti-angiogenic and anti-cancer efficacy.

[0098] Avastin (Bevacizumab) is an anti-VEGF-A monoclonal antibody approved for anticancer and anti-angiogenic treatment. (Garcia et.al Bevacizumab (Avastin®) in cancer treatment: A review of 15 years of clinical experience and future outlook. Cancer T reatment Reviews. Volume 86, June 2020, 102017). Herceptin is a therapeutic antibodythat targets HER-2 in breast cancer and combined with chemotherapy improves survival in HER-2 positive breast cancer. However, resistance develops to Herceptin in patients. (Nahta R and Esteva FJ Herceptin: mechanisms of action and resistance Cancer Letters 232:2 123-138 (2006)). Kevzara (Sarilumab) is a monoclonal antibody that binds to soluble and membrane bound interleukin-6 (IL-6) receptors. Kevzara is approved by the FDA for use in rheumatoid arthritis and has recently shown promise in treating an eye disease, namely posterior segment noninfectious uveitis caused by inflammation of the optic nerve. (Heissigerova et.al. Efficacy and Safety of Sarilumab for the Treatment of Posterior Segment Noninfectious Uveitis (SARIL-NIU) Opthalmology 126:3, 428-437 (2019)) IL6 is found at high concentrations and is deregulated in many cancers, where it promotes tumorigenesis and protects cancer cells from DNA damage induced by cancer treatments. Therefore, blocking or inhibiting IL6 is considered a potential strategy to treat cancers where IL6 signaling is dominant. (Kumari et.al., Role of interleukin-6 in cancer progression and therapeutic resistance. Tumour Biol. (2016) 37(9): 11553) IL6 plays a role in the development of many retinal diseases including age-related macular degeneration, diabetic retinopathy and retinal vein occlusion. Drugs that target IL6 or IL6 receptor could improve treatment of retinal diseases. (Xiao et.al., Interleukin-6 in retinal diseases: From pathogenesis to therapy. Experimental Eye Research (2023) 233: 109556)

[0099] The present invention may further comprise a third binding peptide comprising an endostatin derived peptide sequence having endostatin activity or a plasminogen derived peptide sequence having plasminogen activity or a hepatocyte growth factor derived peptide having hepatocyte growth factor antagonist activity or a vasostatin derived peptide having vasostatin activity, operatively linked to the C-terminus of the heavy chain polypeptide.

[0100] Molecules that inhibit angiogenesis can inhibit cancer growth and metastasis as well as treat eye diseases caused by ocular angiogenesis.

[0101] Endostatin is 20 kDa carboxyl-terminal fragment of type XVIII collagen that is present in walls and basement membranes of blood vessels and plays an important role in endothelial cell adhesion and cytoskeletal organization. Endogenous endostatin inhibits migration and induces apoptosis in endothelial cells, inhibits tumor growth, andimpairs blood vessel maturation in wound healing. It is thought to interfere with the proangiogenic action of growth factors such as basic fibroblast growth factor and VEGF, and is known to inhibit at least 65 different tumor types. Additionally, a study measuring changes in gene expression in human dermal microvascular cells following treatment with endostatin demonstrated a downregulation of several proangiogenic pathways as well as the upregulation of many antiangiogenic genes.

[0102] Endostatin (trade name Endostar) is novel recombinant human endostatin, a protein that specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth. In 2005, Endostar was approved by China’s State Food and Drug Administration (SFDA) for the treatment of non-small cell line lung cancer. As such it has had a long history of human use, but has not been approved in the US or Europe.

[0103] The half-life of the protein in the body is only 10 hours. As such Endostar is administered intravenously once per day over four hours over 14 days. This pharmacokinetic profile unavoidably causes inconvenience, affects quality of life and may reduce patient compliance.

[0104] A more active form of Endostar (P125A) has been identified. In addition, an N-terminal fragment of Endostatin was reported to possess a significant portion of the activity of Endostatin. With wild type Endostatin it has been reported the majority of the effects of endostatin can be ascribed to the N-terminal sequence (1-27) (Sjin RMTT, Satchi-Fainaro R, Birsner AE, Ramanujam VMS, Folkman J, Javaherian K. A 27-amino-acid synthetic peptide corresponding to the NH 2-terminal zinc-binding domain of endostatin is responsible for its antitumor activity. Cancer Res. 2005;65(9):3656-3663. doi: 10.1158 / 0008-5472. CAN-04-1833).

[0105] Endostatin (P125A) has been fused to the C-terminus of other proteins such as Herceptin, an approved drug to block growth factor signals, which improved the activity of the original Herceptin (Shin SU, Cho HM, Merchan J, et al. Targeted delivery of an antibody-mutant human endostatin fusion protein results in enhanced antitumor efficacy. Mol Cancer Then 2011;10(4):603-614. doi:10.1158 / 1535-7163.MCT-10-0804).

[0106] Plasminogen is an endogenous protein that can be cleaved by urokinase activated plasmin and contains five kringle (K) domains of ~80 residues each. Endogenous angiostatin is a 38 kDa amino-terminal fragment of plasminogen, andstudies using recombinant angiostatin demonstrated tumor inhibitory activity resides in domains K1-38. Angiostatin was originally isolated from tumor bearing mice, 6 and has both potent antiangiogenic activity and antiproliferative activity toward endothelial and cancer cells.7 Larger fragments of plasminogen that contain all 5 kringle domains (K1-5) may be even more active than angiostatin alone (K1-3) (Cao R, Wu HL, Veitonmaki N, et al. Suppression of angiogenesis and tumor growth by the inhibitor K1-5 generated by plasmin-mediated proteolysis. Proc Natl Acad Sci U S A. 1999;96(10):5728-5733. doi: 10.1073 / pnas.96.10.5728).

[0107] Recent evidence supports dual antitumor mechanisms for plasminogen derivatives, one affecting angiogenesis and another targeting tumor cells directly. (Sun Q, Xu Q, Dong X, et al. A hybrid protein comprising ATF domain of pro-UK and VAS, an angiogenesis inhibitor, is a potent candidate for targeted cancer therapy. Int J cancer.2008; 123(4):942-950. doi: 10.1002 / I JC.23537).

[0108] Kringle 5 (K5), like angiostatin, is a byproduct of the proteolytic cleavage of plasminogen. In a recent study, Ansell et al., demonstrated K5 functions as a competitive antagonist of hepatocyte growth factor (HGF). (Cho HM, Rosenblatt JD, Kang YS, et al. Enhanced inhibition of murine tumor and human breast tumor xenografts using targeted delivery of an antibody-endostatin fusion protein. Mol Cancer Ther. 2005;4(6):956-967. doi: 10.1158 / 1535-7163. MCT-04-0321 ).

[0109] HGF contains kringle motifs and promotes angiogenesis by stimulating the tyrosine kinase receptor Met. In addition to its potent angiostatic role, K5 can direct a potent antitumor response with its ability to recruit tumor-associated neutrophils and Natural Killer T cells.

[0110] Thus plasminogen has multiple kringle domains which appear to be active alone (angiostatin (K1-4) and K5) and in combination. Thus use of plasminogen derived sequences, in particular the K5 domain with other anti-cancer proteins has been reported (Wang H, Yang Z, Gu J. Therapeutic targeting of angiogenesis with a recombinant CTT peptide-endostatin mimic-kringle 5 protein. Mol Cancer Ther. 2014;13(11):2674-2687. doi: 10.1158 / 1535-7163. MCT-14-0266).

[0111] HGF also factor plays a role in cancer metastasis. NK1 consists of the N-terminal of the first kringle domain of HGF with a Y124A mutation. NK1 (Y124A) binds theHGF receptor MET but does not have agonistic activity. Therefore, NK1 (Y124A) is an HGF antagonist and has a promising role in cancer therapeutics. (Youles et.al. Engineering the NK1 Fragment of Hepatocyte Growth Factor / Scatter Factor as a MET Receptor Antagonist Journal of Molecular Biology 377:3, 616-622 (2008)).

[0112] Vasostatin-1 is a peptide derived from chromogranin A that suppresses angiogenesis, tumor growth and has recently been found to play a role in atherosclerosis. (Pike et.al. Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth J Exp Med 1998 Dec 21 ; 188(12):2349-56 and Sato et.al. Inhibitory effects of vasostatin-1 against atherogenesis Clin Sci (Lond) (2018) 132 (23): 2493-2507)

[0113] The third binding peptide may be selected from the group consisting of a fragment of an endostatin having endostatin activity, an endostatin, a fragment of a plasminogen having plasminogen activity, an endostatin with a P125 mutation, a kringle domain of plasminogen, kringle domains 1 to 5 of plasminogen, kringle domain 5 from a plasminogen, NK1 domain of hepatocyte growth factor with a Y124A mutation or vasostatin 1.

[0114] Arresten is an angiogenesis inhibitor derived from the non-collagenous domain of collagen IV a1 chain. Aikio et.al. demonstrated that arresten inhibited migration and tumor growth in a mouse tumor xenograft mode. This study also found a novel role for arresten in oral squamous carcinoma cell proliferation, survival, motility and invasion. (Aikio et.al. Arresten, a collagen-derived angiogenesis inhibitor, suppresses invasion of squamous cell carcinoma. PLoS One 2012;7(12):e51044).

[0115] NK cells can be activated by CD16, IL15 and ULBP2. CD16 is an NK cell activating receptor that binds the Fc domain of IgGs and drives antibody-dependent cell-mediated cytotoxicity (ADCC). After activation, NK cells release cytotoxic granules that directly lyse tumor cells. (Meng et.al. Leveraging CD16 fusion receptors to remodel the immune response for enhancing anti-tumor immunotherapy in iPSC-derived NK cells. Journal of Hematology & Oncology volume 16, Article number: 62 (2023)). Bispecific antibodies with a first domain of CD16 vhh and a second binding domain of a vhh to a tumor antigen have been used to reduce tumor growth in a mouse model (CN112679609A). CD16-mediated activation of NK cells is a potent signal for inducing ADCC when using these antibodies. Single domain antibody fragments (VHH) comprisean antigen binding domain of the heavy chain without associated light chains. The first known VHH was isolated from camelids (Hamers-Casterman et al., Naturally occurring antibodies devoid of light chains. Nature 363:446-8 (1993)). CD16-specific VHH antibody sequences were disclosed in CN112679609A and US 9,598.499 B2.

[0116] IL-15 is a cytokine that activates NK cells. IL-15 therapies have been tested in several clinical trials in solid cancer, hematologic malignancies and metastasis. (Chu J, Gao F, Yan M, Zhao S, Yan Z, Shi B & Liu Y Natural killer cells: a promising immunotherapy for cancer. Journal of Translational Medicine volume 20, Article number: 240 (2022)). Finally, ULBP2 is a ligand of the NKG2D receptor which is an activating receptor for innate immune responses. In mice, ectopic expression of ULBP2 exhibits an anti-tumor response by recruiting NK cells and T-cells to the tumor. Further, IL15 may help to enhance the immune response against ULBP-expressing tumors by preventing the downregulation of NKG2D. (Sutherland S. et.al. ULBPs, human ligands of the NKG2D receptor, stimulate tumor immunity with enhancement by IL-15. Blood 108:4, 1313-1319 (2006)). The IL15 N72D mutant exhibits superagonist activity which is preserved when linked to a single-chain T-cell receptor domain to generate a tumor-specific fusion protein. (Zhu et.al. Novel Human Interleukin-15 Agonists J. Immunol 183:6, 3598 (2009)).

[0117] The compounds may exhibit may anti-cancer activity. Thus, also taught are methods of treating cancer comprising administration of a composition comprising the compound of the invention, and the use of the compounds for treatment of cancer. The cancer may be a solid cancer. The methods may inhibit metastasis. The method or use may further comprise one or more of the following a) administering chemotherapy; b) administering radiation therapy; or c) administering one or more additional therapeutic agents.

[0118] The compounds may exhibit anti-angiogenic activity to treat eye diseases caused by ocular angiogenesis. Thus, also taught are methods of treating eye diseases caused by ocular angiogenesis comprising administration of a composition comprising the compound of the invention, and the use of the compounds for treatment of eye diseases caused by ocular angiogenesis.

[0119] Ocular angiogenesis or pathological neovascularization is the formation of new blood vessels in the eye that can lead to vision loss in many eye diseases.Neovascularization can occur in response to pathological hypoxia in neovascular age-related macular degeneration (NAMD), diabetic retinopathy (DR), retinal vein occlusion (RVO), retinopathy of prematurity (ROP), neovascular glaucoma, chorodial neovascularization, pathogenic myopia or uveitis. The neovascularization can occur in the cornea or retinal vasculature. For example, the cornea is normally avascular and clear and pathological neovascularization in the cornea can result in vision loss. Anti-angiogenic therapies such as inhibition of vascular endothelial growth factor A (VEGF-A) with anti-VEGF antibody have decreased vision loss in patients with diseases arising from ocular angiogenesis. One of the first anti-VEGF antibodies approved for use to prevent NAMD was bevacizumab which improved loss of vision in clinical trials and was subsequently approved by the FDA in 2004. (Chen et.al. Antiangiogenic therapy for ocular diseases: Current status and challenges. MedComm Future Medicine 2: 1 , e33 (2023) and Kim LA and D’Amore PA A Brief History of Anti-VEGF for the Treatment of Ocular Angiogenesis The American Journal of Pathology 181:2, 376-379 (2012)).

[0120] Figure 1A provides a general schematic of the constructs which can include peptide ligands (Z and Y) operatively linked to the N-terminus of a heavy (HC) and / or a light chain (LC) of an anti-cancer or anti-angiogenic antibody and further anti-angiogenic binding peptides operatively linked to the C-terminal of the heavy chain of the anti-cancer or anti-angiogenic antibody (X). Figure 1B provides an example of a construct where a C20 ligand is bound to the N-terminal of an anti-cancer or anti-angiogenic antibody heavy chain (HC) and K5 peptide bound to the C-terminal of anti-cancer or anti-angiogenic antibody HC.

[0121] The terms “polypeptide”, “peptide”, and “protein” are typically used interchangeably herein to refer to a polymer of amino acid residues. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Each protein or polypeptide will have a unique function. The invention includes polypeptides and functional fragments thereof, as well as mutants and variants having the same biological function or activity.

[0122] In some embodiments, polymeric molecules (e.g., a polypeptide sequence or nucleic acid sequence) are considered to be “homologous” to one another if theirsequences are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical.

[0123] In some embodiments a fragment of a nucleic acid sequence is a fragment of an open reading frame sequence. In some embodiments, such a fragment encodes a polypeptide fragment (as defined herein) of the protein encoded by the open reading frame nucleotide sequence.

[0124] The term “nucleic acid fragment” as used herein refers to a nucleic acid sequence that has a deletion. In some embodiments a fragment of a nucleic acid sequence is a fragment of an open reading frame sequence. In some embodiments, such a fragment encodes a polypeptide fragment (as defined herein) of the protein encoded by the open reading frame nucleotide sequence.

[0125] The term "construct" refers to a nucleic acid sequence encoding a protein, operably operatively linked to a promoter and / or other regulatory sequences.

[0126] The term "genomic sequence" refers to a sequence having non-contiguous open reading frames, where introns interrupt the protein coding regions.

[0127] As used herein, the terms "encoding", "coding", or "encoded" when used in the context of a specified nucleic acid mean that the nucleic acid may be the requisite information to guide translation of the nucleotide sequence into a specified protein. The information by which a protein is encoded is specified by the use of codons. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., as in cDNA).

[0128] The term “percent sequence identity” or “identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. For instance, polynucleotide sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993).

[0129] The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 80%, 85%, or at least about90%, or at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as BLAST, as discussed above.

[0130] As used herein, “heterologous nucleic acid sequence” is any sequence placed at a location in the genome where it does not normally occur.

[0131] A particular nucleic acid sequence also encompasses conservatively modified variants thereof (such as degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Thus, a nucleic acid sequence encoding a protein sequence disclosed herein also encompasses modified variants thereof as described herein. Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art.

[0132] “Operatively linked” or “operably linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with coding sequences of interest to control expression of the coding sequences of interest, as well as expression control sequences that act in trans or at a distance to control expression of the coding sequence.

[0133] A “coding sequence” or “open reading frame” is a sequence of nucleotides that encodes a polypeptide or protein. The termini of the coding sequence are a start codon and a stop codon. The disclosure also includes native, isolated, or recombinant nucleic acid sequences encoding a protein, as well as vectors and / or (host) cells containing the coding sequences for the protein.

[0134] The terms "Fc domain", “Fc peptide” or "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the carboxyl-term inus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al, Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. A "subunit" of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable selfassociation. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0135] Fused or linked means that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0136] Fragments and variants of the disclosed nucleotide sequences and proteins encoded thereby are also encompassed by the present invention. By "fragment1a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby is intended. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein. Accordingly, the present disclosure relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences encoded thereby.

[0137] All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0138] The foregoing description and certain representative embodiments and details of the invention have been presented for purposes of illustration and description of the invention. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. It will be apparent to practitioners skilled in this art that modifications and variations may be made therein without departing from the scope of the invention.ExamplesExample 1 - Constructing C20H-Herceptin-K5

[0139] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to C20 bound to Herceptin heavy chain bound to a constant heavy (CH) chain domain of Human lgG1bound to a linker bound to K5. The light chain sequence has a signal peptide bound to Herceptin light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 1 and the light chain sequence is set out in SEQ ID. No. 2, below.

[0140] Heavy chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTEVQLVESGGGLVQPGGSLRLSC AASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYL QMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSSEEDCMFGNGKGYR GKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTA NPRKLYDYCDVPQCAA

[0141] Light chain:MHSSALLCCLVLLTGVRADIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFG QGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0142] Purity was > 85% as determined by reducing SDS-PAGE and >95% by nonreducing SDS-PAGE. Purity was >97% as determined by SEC-HPLC at 280 nm. Endotoxin: 0-1 X EU / mg. Concentration was 1.02 mg / mL. The formulation buffer was PBS.

[0143] As shown in Figure 2, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 2 - Constructing C20L-Herceptin-K5

[0144] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to Herceptin heavy chain bound to a constant heavy (CH) chain domain of Human lgG1 bound to a linker bound to K5. The light chain sequence has a signal peptide bound to C20 bound to Herceptin light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 3 and the light chain sequence is set out in SEQ ID No. 4, below.

[0145] Heavy chain:MHSSALLCCLVLLTGVRAEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA PGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGKGGGGSGGGGSGGGGSSEEDCMFGNGKGYRGKRATTVTGTPCQDWA AQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTANPRKLYDYCDVPQCAA

[0146] Light chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPE DFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC

[0147] Purity was > <50% as determined by reducing SDS-PAGE and >95% as determined by non-reducing SDS-PAGE. Purity was >98% as determined by SEC-HPLC at 280 nm. Endotoxin: 0-1 Ell / rng. Concentration was 0.91 mg / mL.

[0148] As shown in Figure 3, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 3 - Constructing C20HL-Herceptin-K5

[0149] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has signal peptide bound to C20 bound to Herceptin heavy chain bound to a constant heavy (CH) chain domain of Human lgG1 bound to a linker bound to K5. The light chain sequence has a signal peptide bound to C20 bound to Herceptin light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 1 and the light chain sequence is set out in SEQ ID. No. 4, below.

[0150] Heavy chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTEVQLVESGGGLVQPGGSLRLSC AASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYL QMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSSEEDCMFGNGKGYR GKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTA NPRKLYDYCDVPQCAA

[0151] Light chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTDIQMTQSPSSLSASVGDRVTITC RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPE DFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC

[0152] Purity was <50% as determined by reducing SDS-PAGE and >95% by nonreducing SDS-PAGE. Purity was >97% as determined by SEC-HPLC at 280 nm. Endotoxin: 1-5 Ell / rng. Concentration was 0.35 mg / mL.

[0153] As shown in Figure 4, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 4 - Constructing Tetravalent C20-Fc4

[0154] This fusion protein has two heavy chains polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to C20 bound to a linker bound to a constant heavy (CH) chain domain of Human lgG4 with mutation S228P. The light chain sequence has a signal peptide bound to C20 bound to a linker bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified byProtein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 5 and the light chain sequence is set out in SEQ ID No. 6, below.

[0155] Heavy chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTGGGSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGK

[0156] Light chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTGGGSRTVAAPSVFIFPPSDEQLK SGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0157] Purity was >75% as determined by reducing SDS-PAGE and >95% by nonreducing SDS-PAGE. Purity was >94% as determined by SEC-HPLC at 280 nm. Endotoxin: 0-1 EU / rng. Concentration was 0.84 mg / mL.

[0158] As shown in Figure 5, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate as about 35-50 kDa and 15-20 kDa, respectively. Non-reduced protein migrates about 100-150 kDa.Example 5 - Constructing C20H-Kevzara-K5

[0159] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to C20 bound to Kevzara heavy chain bound to a constant heavy (CH) chain domain of Human lgG1 with a mutation (in the QVTYLPPSRDEL area of the heavy chain) bound to a linker bound to K5. The light chain sequence has a signal peptide bound to Kevzara light chain bound toa constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 7 and the light chain sequence is set out in SEQ ID. No. 8, below.

[0160] Heavy chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTEVQLVESGGGLVQPGRSLRLSC AASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSL FLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTP EVTCVWD VS H E D P E VKF N WYVDGVE VH N AKTKP RE EQYN STYRVVSVLTVLH Q DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTYLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSSEEDCMFGNGKGYRGKRA TTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTANPRK LYDYCDVPQCAA

[0161] Light chain:MHSSALLCCLVLLTGVRADIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP GKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQQANSFPYTFG QGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0162] Purity was <50% as determined by reducing SDS-PAGE and >85% by nonreducing SDS-PAGE. Purity was >90% as determined by SEC-HPLC at 280 nm. Endotoxin: 0-1 EU / mg. Concentration was 1.97 mg / mL.

[0163] As shown in Figure 6, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain andLight chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 6 - Constructing Praluent Control

[0164] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to Praluent heavy chain bound to constant heavy (CH) chain domain of Human lgG1. The light chain sequence has a signal peptide bound to light chain Praluent bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 9 and the light chain sequence is set out in SEQ ID. No. 10, below.

[0165] Heavy chain:MHSSALLCCLVLLTGVRAEVQLVESGGGLVQPGGSLRLSCAASGFTFNNYAMNWVR QAPGKGLDWVSTISGSGGTTNYADSVKGRFIISRDSSKHTLYLQMNSLRAEDTAVYYC AKDSNWGNFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG

[0166] Light chain:MHSSALLCCLVLLTGVRADIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNRNFLG WYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY YTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

[0167] Purity was >95% as determined by reducing SDS-PAGE and >95% by nonreducing SDS-PAGE. Purity was >98% as determined by SEC-HPLC at 280 nm. Endotoxin: <1 Ell / rng. Concentration was 0.67 mg / mL.

[0168] As shown in Figure 7, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 7 - Constructing Praluent-Endo(P125A)

[0169] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to Praluent heavy chain bound to constant heavy (CH) chain domain of Human lgG1 with E356D M358L mutations bound to a linker bound to endostatin with a proline to alanine mutation (P125A-endostatin). The light chain sequence has a signal peptide bound to light chain Praluent bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 11 and the light chain sequence is set out in SEQ ID. No. 10, below.

[0170] Heavy chain:MHSSALLCCLVLLTGVRAEVQLVESGGGLVQPGGSLRLSCAASGFTFNNYAMNWVR QAPGKGLDWVSTISGSGGTTNYADSVKGRFIISRDSSKHTLYLQMNSLRAEDTAVYYC AKDSNWGNFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGGGGSHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTF RAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDG KDVLRHPTWPQKSVWHGSDANGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQS AASC H H AYI VLC I E N S FMTAS K

[0171] Light chain:MHSSALLCCLVLLTGVRADIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNRNFLG WYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY YTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

[0172] Purity was >95% as determined by reducing SDS-PAGE and >95% by nonreducing SDS-PAGE. Purity was >98% as determined by SEC-HPLC at 280 nm. Endotoxin: <1 Ell / rng. Concentration was 0.77 mg / mL.

[0173] As shown in Figure 8, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 8 - Constructing C20-Praluent-Endo(P125A)

[0174] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain has a signal peptide bound to C20 bound to Praluent heavy chain bound to constant heavy (CH) chain domain of Human lgG1 with E356D M358L mutations bound to a linker bound to endostatin with a proline to alanine mutation (P125A-endostatin). The light chain sequence has a signal peptide bound to light chain Praluent bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (ifneeded), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 12 and the light chain sequence is set out in SEQ ID. No. 10, below.

[0175] Heavy chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTEVQLVESGGGLVQPGGSLRLSC AASGFTFNNYAMNWVRQAPGKGLDWVSTISGSGGTTNYADSVKGRFIISRDSSKHTL YLQMNSLRAEDTAVYYCAKDSNWGNFDLWGRGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGGGGSHSHRDFQPVLHLVALNSPLSGGMRGIRGAD FQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDELLFPSWEALFSG SEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDANGRRLTESYCETWRTEAPSA TGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK

[0176] Light chain:MHSSALLCCLVLLTGVRADIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNRNFLG WYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY YTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

[0177] Purity was >95% as determined by reducing SDS-PAGE and >95% by nonreducing SDS-PAGE. Purity was >96% as determined by SEC-HPLC at 280 nm. Endotoxin: <1 EU / rng. Concentration was 0.8 mg / mL.

[0178] As shown in Figure 9, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 70-100 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 9 - Constructing C20-Praluent

[0179] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence of a signal peptide bound to C20 bound to Praluent heavy chain bound to constant heavy (CH) chain domain of Human lgG1 with E356D M358L mutations. The light chain sequence has a signal peptide bound to light chain Praluent bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 13 and the light chain sequence is set out in SEQ ID. No. 10, below.

[0180] Heavy chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTEVQLVESGGGLVQPGGSLRLSC AASGFTFNNYAMNWVRQAPGKGLDWVSTISGSGGTTNYADSVKGRFIISRDSSKHTL YLQMNSLRAEDTAVYYCAKDSNWGNFDLWGRGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPG

[0181] Light chain:MHSSALLCCLVLLTGVRADIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNRNFLG WYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY YTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

[0182] Purity was >95% as determined by reducing SDS-PAGE and >95% by nonreducing SDS-PAGE. Purity was 100% as determined by SEC-HPLC at 280 nm.Endotoxin: <1 Ell / mg. Concentration was 0.95 mg / mL.

[0183] As shown in Figure 10, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 10- In Vivo Anti-Cancer Testing Using A Zebrafish Model

[0184] The purpose of this study was to evaluate the anti-tumor efficacy of compounds on primary tumor growth and metastasis of the HCT 116 cell line in a zebrafish model.Zebrafish tumor xenograft (ZTX) model

[0185] The zebrafish model is a short but predictive model to screen anti-cancer agents that has gained wide acceptance as a useful in vivo model.

[0186] Transgenic Tg(fli1 :EGFP)y1zebrafish embryos were raised at 28°C for 48 hours in E3 embryo medium 0.2 mM 1-Phenyl-2 -Thiourea aka PTU. Unfertilized eggs or larvae that did not appear healthy or exhibited any obvious developmental defects were excluded before experimental onset.

[0187] The efficacy evaluation of compounds was performed using HCT116 cells. HCT116 cell line was obtained from ATCC and culturing was performed following the providers instructions. HCT116 cancer cells were labeled with Dil red fluorescent dye. Approximately 700 Dil-labeled cancer cells (red) were subcutaneously co-implanted into the perivitelline space of 2 days old larvae ± compounds. The HCT-116 cell line was chosen due to its high expression of PCSK9 and sensitivity to uPAR (PMID: 25113506 and 19484792).

[0188] Larvae in which tumor cells had been inadvertently injected into circulation or larvae with erroneous implantation of the tumor in the yolk rather than the perivitelline space were excluded from the study. Selected tumor-bearing embryos were sorted into experimental groups (20 embryos / group), and pictures of primary tumors were taken right after injection. Tumor-bearing embryos were incubated in E3 / PTU medium for 72 hoursat 35.5°C. After incubation, pictures of the primary tumors were taken by using a red fluorescent filter. Larvae that died or were lost by other means during the study were excluded from the final analysis.Analysis of tumor growth inhibition and metastasis

[0189] Anti-cancer efficacy of the constructs was determined as the change in primary tumor size (i.e. tumor growth or reduction) and the number of disseminated cancer cells to the caudal venous plexus (CVP) three days after implantation.

[0190] Images obtained right after implantation (day 0) and after 72 hours incubation (day 3) were analyzed by using in-house developed software. Primary tumor regression was calculated and normalized to the negative treatment group, and the number of disseminated cells to the CVP after 3 days were counted manually.Statistical analysis

[0191] Data are shown as mean ± SEM. D’Agostino & Pearson normality test was performed. A one-way ANOVA was performed and followed by two-tailed Student’s t-test. Definitive outliers were detected using the ROUT method with a Q=0.1%. Graphs and experimental data were obtained and analyzed by using GraphPad Prism v10.1.2.Constructs tested in the zebrafish tumor xenograft model

[0192] Avastin antibody was used as a positive control. Table 1 shows the experimental groups tested. The compound ScFv-ATN658-ABD required human albumin. Due to the low concentration of the compound stock (0.7 mg / mL), the tumor cells were resuspended in the stock solution for tumor implantation, and human albumin was injected intratumorally (ITU) right after tumor implantation.Table 1. Description of groups for efficacy evaluation of compounds using HCT116 cellsGroup Evaluation of Evaluation of Treatment Cone. Admin.size primary tumor metastasis Negative control - 20 - 0 and 72 hours 72 hours A vastin 2 mg / mL 20 co-injection 0 and 72 hours 72 hours ATF-Fc(IgG4)- 2 mg / mL 20 co-injection 0 and 72 hours 72 hours Endostatin (P125A)ATF-Fc (IgG4)-K5 2 mg / mL 20 co-injection 0 and 72 hours 72 hours ScFv-ATN658-ABD 0.7 mg / mL co-injectiont 4 20 0 and 72 hours 72 hours human albumin 10 mg / mL ITU injectionBivalent C20-Fc 1.6 mg / mL 20 co-injection 0 and 72 hours 72 hours Bivalent C20-Fc 0.67 mg / mL 20 co-injection 0 and 72 hours 72 hours Praluent 0.67 mg / mL 20 co-injection 0 and 72 hours 72 hours C20-Praluent 0.67 mg / mL 20 co-injection 0 and 72 hours 72 hours ATF-Endo(P 125A)-Fc1 mg / mL 20 co-injection 0 and 72 hours 72 hours (IgG4)ATF-Arrestin-Fc (IgG4) 1 mg / mL 20 co-injection 0 and 72 hours 72 hoursATF-Fc 1 mg / mL 20 co-injection 0 and 72 hours 72 hours ResultsPrimary tumor evaluationATF compounds

[0193] The efficacy evaluation of ATF compounds on primary tumor size after 72 hours post-tumor implantation is shown in Figure 11. Normalized data with respect to the negative control group is shown. Data are presented as mean ± SEM; a two-tailed Student’s t-test was performed. A significantly smaller primary tumor size with ATF-Arrestin-Fc4 1 mg / mL treatment compared to ATF-Fc4-Endo 2 mg / mL treatment was observed.

[0194] Borderline significance was observed between ATF-Fc4-Endo and ATF-Fc4- K5 treatments (p=0.078) and ATF-Arrestin-Fc4 and ATF-Fc1 treatments (p=0.063).C20 and Praluent constructs

[0195] The efficacy evaluation of C20 and Praluent compounds on primary tumor size after 72 hours post-tumor implantation is shown in Figure 12. Normalized data with respect to the negative control group is shown. Data are presented as mean ± SEM; a two-tailed Student’s t-test was performed. A borderline-significant smaller primary tumorsize was observed with C20-Praluent 0.67 mg / mL treatment compared to Bivalent C20-Fc 0.67 mg / mL treatment (p=0.0586).

[0196] Any other comparison between treatments exhibited a non-significant difference in primary tumor size after 3 days.All constructs

[0197] The efficacy evaluation of all compounds on primary tumor size after 72 hours post-tumor implantation is shown in Figure 13. Normalized data with respect to the negative control group is shown. Data are presented as mean ± SEM; One-way ANOVA was performed (p=0.0763) followed by a two-tailed Student’s t-test, *p<0.05. Compounds ATF-Fc4-K5 2 mg / mL, Praluent 0.67 mg / mL, C20-Praluent 0.67 mg / mL, and ATF-Arrestin-Fc4 1 mg / mL significantly decreased the primary tumor size compared to the control group after 3 days of treatment.

[0198] The Bivalent C20-Fc compound at 1.6 mg / mL decreased the primary tumor size compared to the control group; however, this effect did not reach a mathematical significance and showed a borderline significance with a p-value of 0.093 (Figure 13).Metastasis

[0199] The efficacy evaluation of compounds on metastasis of HCT116 cells after 72 hours post-tumor implantation is shown in Figure 14. Average number of disseminated cells to the CVP is shown. Data are presented as mean ± SEM; One-way ANOVA was performed (p=0.1134) followed by a two-tailed Student’s t-test. A reduction in the number of disseminated tumor cells to the CVP was observed with the bivalent-C20 Fc treatment compared to the control group; however, this effect did not reach a significance (Figure 14).

[0200] Figure 14 also shows that treatment with the different compounds did not affect the metastatic dissemination of HCT116 cells to the caudal vein plexus after 3 days of treatment. The positive control, Avastin, did not affect the dissemination of tumor cells to the CVP.Conclusions

[0201] The compounds ATF-Fc4-K5, Praluent, C20-Praluent, and ATF-Arrestin-Fc4have an anti-tumor effect on HCT116 xenografts in vivo in the zebrafish model. The ATF-Arrestin-Fc4 construct had the strongest effect on primary tumor regression.

[0202] Different construct configurations, such as ATF-Fc4-Endostatin and ATF-Endostatin-Fc4, do not have anti-tumor efficacy on HCT116 cells at the tested concentrations. Due to the difference in concentration, it is difficult to make a comparison of the anti-tumor effect for these two constructs.

[0203] The Praulent compound has anti-tumor activity on HCT116 xenografts and this effect is slightly increased when combined with the C20 motif.

[0204] Avastin control has no anti-tumoral or anti-metastasis effect on HCT116 xenografts at the tested concentration; this might be due to the presence of other pro-angiogenic factors that could mask the effect of Avastin and contribute to tumor cell dissemination.Example 11 - In Vivo Anti-Cancer Testing Using A Zebrafish Model of C20-Herceptin and C20-Kevzara ConstructsZebrafish tumor xenograft model

[0205] To test the C20-Herceptin and C20-Kevzara constructs, the zebrafish tumor xenograft (ZTX) model from Example 10 was used with the modification that DU 145 cancer cells were used to create the xenograft, rather than HCT116 cells.Constructs tested in the zebrafish tumor xenograft model

[0206] The constructs tested were Herceptin at 2 mg / mL, Herceptin-K5 at 0.9 mg / mL, C20H-Herceptin-K5 at 0.9 mg / mL, C20L-Herceptin-K5 at 0.9 mg / mL, C20H-Kevzara-K5 at 0.9 mg / mL, C20-tetravalent at 0.8 mg / mL and C20-bivalent at 0.8 mg / mL.ResultsPrimary tumor evaluation

[0207] The efficacy evaluation of all constructs on primary tumor size is shown in Figure 15. Normalized data with respect to the negative control group is shown. Data are presented as mean ± SEM; a two-tailed Student’s t-test was performed. Normality testswere performed followed by parametric or non-parametric tests such as One-way ANOVA followed by two-tailed Student’s t-test. A significantly smaller primary tumor size compared to control was observed with Herceptin, Herceptin-K5, C20H-Herceptin-K5, C20L-Herceptin-K5 and C20H-Kevzara-K5.Example 12 - Constructing L17H-C20L-Avastin-Endo

[0208] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to L17 bound to a linker bound to heavy chain Avastin bound to constant heavy (CH) chain domain of Human IgG 1 with N297A mutations bound to a linker bound to endostatin. The light chain sequence has a signal peptide bound to C20 bound to light chain Avastin bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 14 and the light chain sequence is set out in SEQ ID No. 15, below.

[0209] Heavy chain:MHSSALLCCLVLLTGVRAMGAQTNFMPMDDLEQRLYEQFILQQGLEGGGSEVQLVES GGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADS VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSH SHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLY SIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDANGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCI ENSFMTASK

[0210] Light chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTGGGSDIQMTQSPSSLSASVGDR VTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECExample 13 - Constructing L17H-C20L-Avastin-K5

[0211] This fusion protein has two heavy chains and two light chains. The heavy chain sequence has a signal peptide bound to L17 bound to a linker bound to heavy chain Avastin bound to constant heavy (CH) chain domain of Human lgG1 with N297A mutations bound to a linker bound to K5. The light chain sequence has a signal peptide bound to C20 bound to a linker bound to light chain Avastin bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 16 and the light chain sequence is set out in SEQ ID. No. 15, below.

[0212] Heavy chain:MHSSALLCCLVLLTGVRAMGAQTNFMPMDDLEQRLYEQFILQQGLEGGGSEVQLVES GGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADS VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSAS TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYASTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSS EEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRN PDGDVGGPWCYTANPRKLYDYCDVPQCAA

[0213] Light chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTGGGSDIQMTQSPSSLSASVGDR VTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGECExample 14 - Constructing L17H-C20L-Kevzara-K5

[0214] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to L17 bound to a linker bound to Kevzara heavy chain bound to constant heavy (CH) chain domain of Human IgG 1 with mutations bound to a linker bound to K5. The light chain sequence has a signal peptide bound to C20 bound to a linker bound to Kevzara light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 17 and the light chain sequence is set out in SEQ ID No. 18, below.

[0215] Heavy chain:MHSSALLCCLVLLTGVRAMGAQTNFMPMDDLEQRLYEQFILQQGLEGGGSEVQLVES GGGLVQPGRSLRLSCAASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADS VKGRFTISRDNAENSLFLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTYLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSSEE DCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPD GDVGGPWCYTANPRKLYDYCDVPQCDEES

[0216] Light chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTGGGSDIQMTQSPSSVSASVGDR VTITCRASQGISSWLAWYQQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISS LQPEDFASYYCQQANSFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC

[0217] Purity was >95% as determined by reducing SDS-PAGE. Purity was > 91% as determined by SEC-HPLC. Endotoxin: 0-1 Ell / rng. Concentration was 1.09 mg / mL.

[0218] As shown in Figure 16, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 15 - Constructing C20L-Kevzara-L17-Endostatin(P125A)

[0219] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to Kevzara heavy chain bound to constant heavy (CH) chain domain of Human lgG1 with mutations bound to a linker bound to L17 bound to endostatin with a proline to alanine mutation (P125A-endostatin). The light chain sequence has a signal peptide bound to C20 bound to Kevzara light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulkwith high purity. The heavy chain sequence is set out in SEQ ID No. 19 and the light chain sequence is set out in SEQ ID No. 20, below.

[0220] Heavy chain:MHSSALLCCLVLLTGVRAEVQLVESGGGLVQPGRSLRLSCAASRFTFDDYAMHWVRQ APGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSLFLQMNGLRAEDTALYYC AKGRDSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVTYLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGKGGGGSMGAQTNFMPMDDLEQRLYEQFILQQGLEGGGSGGGGSHSHRDFQP VLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADR AAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSD ANGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASK

[0221] Light chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTDIQMTQSPSSVSASVGDRVTITC RASQGISSWLAWYQQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQPE DFASYYCQQANSFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC

[0222] Purity was >75% as determined by reducing SDS-PAGE. Purity was >76 % as determined by SEC-HPLC at 280 nm. Endotoxin: 0-1 EU / rng. Concentration was 1.09 mg / mL.

[0223] As shown in Figure 17, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 70-100 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 16 - Constructing C20H-Kevzara-HGF(Y124Y)NK1

[0224] This fusion protein has two heavy chain polypeptides and light chain polypeptides. The heavy chain sequence has a signal peptide bound to C20 bound to a linker bound to Kevzara heavy chain bound to constant heavy (CH) chain domain of Human lgG1 with mutations bound to a linker bound to NK1 fragment of hepatocyte growth factor / scatter factor with Y124Y. The light chain sequence has a signal peptide bound to Kevzara light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 21 and the light chain sequence is set out in SEQ ID. No. 8, below.

[0225] Heavy chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTGGGSEVQLVESGGGLVQPGRSL RLSCAASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNA ENSLFLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSS SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTYLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSMWVTKLLPALLLQHV LLHLLLLPIAIPYAEGQRKRRNTIHEFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRN KGLPFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYENKDAIRNCIIGKGRS YKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRGKDLQENYCRNPRGEEGGPWCFTS NPEVRYEVCDIPQCDEE

[0226] Light chain:MHSSALLCCLVLLTGVRADIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP GKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQQANSFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECExample 17 - Constructing C20-Praulent-Vasostatin1

[0227] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to C20 bound to Praulent heavy chain bound to constant heavy (CH) chain domain of Human lgG1 with E356D M358L mutations bound to a linker bound to vasostatin 1. The light chain sequence has a signal peptide bound to Praulent light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 22 and the light chain sequence is set out in SEQ ID. No. 10, below.

[0228] Heavy chain:MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTEVQLVESGGGLVQPGGSLRLSC AASGFTFNNYAMNWVRQAPGKGLDWVSTISGSGGTTNYADSVKGRFIISRDSSKHTL YLQMNSLRAEDTAVYYCAKDSNWGNFDLWGRGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSLPVNSPMNKGDTEVMKCIVEVISD TLSKPSPMPVSQECFETLRGDERILSILRHQNLLKELQDLALQGAKERAHQQ

[0229] Light chain:MHSSALLCCLVLLTGVRADIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNRNFLG WYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY YTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGECExample 18 - Constructing ATF-Fc4-Endostatin(P125A)-L17H

[0230] This fusion protein has two heavy chain polypeptides. The heavy chain sequence has a signal peptide bound to ATF bound to a linker bound to an Fc of Human lgG4 with S228P mutation bound to a linker bound to endostatin (P125A) bound to L17. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 23, below.MEWSWVFLFFLSVTTGVHSSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKF GGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQ LGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGSGSGSPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGG GSHSHRDFQPVLHLVALNSPLSGGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQ DLYSIVRRADRAAVPIVNLKDELLFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTW PQKSVWHGSDANGRRLTESYCETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYI VLCIENSFMTASKGGGGSMGAQTNFMPMDDLEQRLYEQFILQQGLE

[0231] Purity was >95% as determined by reducing SDS-PAGE. Purity was >88% as determined by SEC-HPLC. Endotoxin: 0-1 EU / rng. Concentration was 0.86 mg / mL. Formulation buffer was PBS at pH 7.2-7.4.

[0232] As shown in Figure 18, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result the reduced protein migrates at about 50-70 kDa. Non-reduced protein migrates about 100-150 kDa.Example 19 - Constructing Kevzara Control

[0233] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to Kevzara heavy chain bound to a constant heavy chain domain of human lgG1 with a mutation. The light chain sequence has a signal peptide bound to Kevzara light chain bound to a constant domain of human kappa light chain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 24 and the light chain sequence is set out in SEQ ID. No. 10, below.

[0234] Heavy chain MHSSALLCCLVLLTGVRAEVQLVESGGGLVQPGRSLRLSCAASRFTFDDYAMHWVRQ APGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSLFLQMNGLRAEDTALYYC AKGRDSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVTYLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK

[0235] Light chain MHSSALLCCLVLLTGVRADIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNRNFLG WYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY YTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

[0236] Purity was >95% as determined by reducing SDS-PAGE. Purity was > 96% as determined by SEC-HPLC. Endotoxin: 0-1 Ell / rng. Concentration was 1.33 mg / mL. Formulation buffer was PBS pH 7.2-7.4.

[0237] As shown in Figure 19, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 20 - Constructing C20-Kevzara

[0238] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to C20 bound to Kevzara heavy chain bound to a constant heavy chain domain of human lgG1 with a mutation. The light chain sequence has a signal peptide bound to Kevzara light chain bound to a constant domain of human kappa light chain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 25 and the light chain sequence is set out in SEQ ID. No. 10, below.

[0239] Heavy chain MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTEVQLVESGGGLVQPGRSLRLSC AASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSL FLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTP EVTCVWD VS H E D P E VKF N WYVDGVE VH N AKTKP RE EQYN STYRVVSVLTVLH Q DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTYLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0240] Light chain MHSSALLCCLVLLTGVRADIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNRNFLG WYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY YTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

[0241] Purity was >95% as determined by reducing SDS-PAGE. Purity was > 95% as determined by SEC-HPLC. Endotoxin: 0-1 Ell / rng. Concentration was 1.41 mg / mL. Formulation buffer was PBS pH 7.2-7.4.

[0242] As shown in Figure 20, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 21 - Constructing C20-Kevzara-K5-L17

[0243] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to C20 bound to Kevzara heavy chain bound to a constant heavy chain domain of human lgG1 with a mutation bound to a linker bound to K5 bound to a linker bound to L17. The light chain sequence has a signal peptide bound to Kevzara light chain bound to a constant domain of human kappa light chain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 26 and the light chain sequence is set out in SEQ ID. No. 10, below.

[0244] Heavy chainMHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTEVQLVESGGGLVQPGRSLRLSCAASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSL FLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTP EVTCVWD VS H E D P E VKF N WYVDGVE VH N AKTKP RE EQYN STYRVVSVLTVLH Q DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTYLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSSEEDCMFGNGKGYRGKRA TTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTANPRK LYDYCDVPQCDEESGGGGSMGAQTNFMPMDDLEQRLYEQFILQQGLE

[0245] Light chain MHSSALLCCLVLLTGVRADIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNRNFLG WYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY YTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

[0246] Purity was >65% as determined by reducing SDS-PAGE. Purity was > 85% as determined by SEC-HPLC. Endotoxin: 0-1 Ell / rng. Concentration was 1.11 mg / mL. Formulation buffer was PBS pH 7.2-7.4.

[0247] As shown in Figure 21 , SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 22 - Constructing C20-Kevzara-Vasostatin1-L17

[0248] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to C20 bound to Kevzara heavy chain bound to a constant heavy chain domain of human lgG1 with a mutation bound to a linker bound to Vasostatin bound to a linker bound to L17. The light chain sequence has a signal peptide bound to Kevzara light chain bound to a constantdomain of human kappa light chain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 27 and the light chain sequence is set out in SEQ ID. No. 10, below .

[0249] Heavy chain MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTEVQLVESGGGLVQPGRSLRLSC AASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSL FLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTP EVTCVWD VS H E D P E VKF N WYVDGVE VH N AKTKP RE EQYN STYRVVSVLTVLH Q DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTYLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSLPVNSPMNKGDTEVMKCIV EVISDTLSKPSPMPVSQECFETLRGDERILSILRHQNLLKELQDLALQGAKERAHQQGG GGSMGAQTNFMPMDDLEQRLYEQFILQQGLE

[0250] Light chain MHSSALLCCLVLLTGVRADIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNRNFLG WYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY YTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGECExample 23 - Constructing C20-Kevzara-Endostatin(p125A)-L17

[0251] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence with a signal peptide bound to C20 bound to Kevzara heavy chain bound to a constant heavy chain domain of human lgG1 with amutation bound to a linker bound to endostatin (p125A) bound to a linker bound to L17. The light chain sequence has a signal peptide bound to Kevzara light chain bound to a constant domain of human kappa light chain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 28 and the light chain sequence is set out in SEQ ID. No. 10, below.

[0252] Heavy chain MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTEVQLVESGGGLVQPGRSLRLSC AASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSL FLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTP EVTCVWD VS H E D P E VKF N WYVDGVE VH N AKTKP RE EQYN STYRVVSVLTVLH Q DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTYLPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSHSHRDFQPVLHLVALNSPLS GGMRGIRGADFQCFQQARAVGLAGTFRAFLSSRLQDLYSIVRRADRAAVPIVNLKDEL LFPSWEALFSGSEGPLKPGARIFSFDGKDVLRHPTWPQKSVWHGSDANGRRLTESYC ETWRTEAPSATGQASSLLGGRLLGQSAASCHHAYIVLCIENSFMTASKGGGGSMGAQ TNFMPMDDLEQRLYEQFILQQGLE

[0253] Light chain MHSSALLCCLVLLTGVRADIVMTQSPDSLAVSLGERATINCKSSQSVLYRSNNRNFLG WYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY YTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGECExample 24 - Constructing C20L-Praluent-Vasostatin1

[0254] This fusion protein has two heavy chain polypeptides and two light chain polypeptides, uses a heavy chain sequence has a signal peptide bound to Praluent heavy chain bound to a constant heavy chain domain of human lgG1 with E356D M358L mutations bound to a linker bound to vasostatin 1. The light chain sequence has a signal peptide bound to C20 bound to Praluent light chain bound to a constant domain of human kappa light chain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 29 and the light chain sequence is set out in SEQ ID No. 30, below.

[0255] Heavy chain MHSSALLCCLVLLTGVRAEVQLVESGGGLVQPGGSLRLSCAASGFTFNNYAMNWVR QAPGKGLDWVSTISGSGGTTNYADSVKGRFIISRDSSKHTLYLQMNSLRAEDTAVYYC AKDSNWGNFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGGGGGSGGGGSGGGGSLPVNSPMNKGDTEVMKCIVEVISDTLSKPSPMPV SQECFETLRGDERILSILRHQNLLKELQDLALQGAKERAHQQ

[0256] Light chain MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTDIVMTQSPDSLAVSLGERATINC KSSQSVLYRSNNRNFLGWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQYYTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASW CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGECExample 25 - Constructing C20L-Praluent

[0257] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to Praluent heavy chain bound to a constant heavy chain domain of human lgG1 with E356D M358L mutations. The light chain sequence has a signal peptide bound to C20 bound to Praluent light chain bound to a constant domain of human kappa light chain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 9 and the light chain sequence is set out in SEQ ID No. 30, below.

[0258] Heavy chain MHSSALLCCLVLLTGVRAEVQLVESGGGLVQPGGSLRLSCAASGFTFNNYAMNWVR QAPGKGLDWVSTISGSGGTTNYADSVKGRFIISRDSSKHTLYLQMNSLRAEDTAVYYC AKDSNWGNFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG

[0259] Light chain MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTDIVMTQSPDSLAVSLGERATINC KSSQSVLYRSNNRNFLGWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCQQYYTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASW CLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGECExample 26 - Constructing C20 Bivalent

[0260] This fusion protein has two heavy chain polypeptides. The heavy chain sequence has a signal peptide bound to C20 bound to a linker bound to an Fc of Human lgG4 with a S228P mutation. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 31, below.MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTGGGGSESKYGPPCPPCPAPEFL GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKExample 27 - Constructing ATF-Fc4-Vasostatin1

[0261] This fusion protein has two heavy chain polypeptides. The heavy chain sequence has a signal peptide bound to ATF bound to a linker bound to an Fc of Human lgG4 with a S228P mutation bound to a linker bound to vasostatin 1. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 32, below.MEWSWVFLFFLSVTTGVHSSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKF GGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQ LGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGSGSGSPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGG GSLPVNSPMNKGDTEVMKCIVEVISDTLSKPSPMPVSQECFETLRGDERILSILRHQNL LKELQDLALQGAKERAHQQExample 28- Constructing L17H-C20L-Herceptin-K5

[0262] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has L17 bound to a linker bound to Herceptin heavy chain bound to constant heavy (CH) chain domain of Human lgG1 with mutations bound to a linker bound to K5. The light chain sequence has to C20 bound to a linker bound to Herceptin light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 33 and the light chain sequence is set out in SEQ ID No. 34, below.

[0263] Heavy chain:MGAQTNFMPMDDLEQRLYEQFILQQGLEGGGSEVQLVESGGGLVQPGRSLRLSCAA SRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYADSVKGRFTISRDNAENSLFL QMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGT AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P EVTC WVDVS H E D P EVKF N WYVDGVEVH N AKTKP RE EQYN STYR WSVLTVLH Q D WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVTYLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSSEEDCMFGNGKGYRGKRATT VTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTANPRKLY DYCDVPQCDEES

[0264] Light chain:AEPMPHSLNFSQYLWYTGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWY QQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTP PTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC

[0265] Purity was >85% as determined by reducing SDS-PAGE. Purity was > 98% as determined by SEC-HPLC. Endotoxin: 0-1 Ell / rng. Concentration was 2.18 mg / mL. Formulation buffer was PBS at pH 7.2-7.4.

[0266] As shown in Figure 22, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 29 - Constructing (C20-L17)H-Herceptin-K5

[0267] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has C20 bound to a linker bound to L17 bound to a linker bound to Herceptin heavy chain bound to constant heavy (CH) chain domain of Human lgG1 with mutations bound to a linker bound to K5. The light chain sequence has Herceptin light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 35 and the light chain sequence is set out in SEQ ID No. 36, below.

[0268] Heavy chain:AEPMPHSLNFSQYLWYTGGGSMGAQTNFMPMDDLEQRLYEQFILQQGLEGGGSEVQ LVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSG GGGSSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEK NYCRNPDGDVGGPWCYTANPRKLYDYCDVPQCAA

[0269] Light chain:DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGV PSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0270] Purity was >85% as determined by reducing SDS-PAGE. Purity was > 99% as determined by SEC-HPLC. Endotoxin: 0-1 Ell / rng. Concentration was 1.08 mg / mL. Formulation buffer was PBS at pH 7.2-7.4.

[0271] As shown in Figure 23, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 30 - Constructing- C20L-Herceptin-L17

[0272] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has Herceptin heavy chain bound to constant heavy (CH) chain domain of Human lgG1 bound to a linker bound to L17. The light chain sequence has C20 bound to a linker bound to Herceptin light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and thensubjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 37 and the light chain sequence is set out in SEQ ID No. 34, below.

[0273] Heavy chain:EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGY TRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGG GSMGAQTNFMPMDDLEQRLYEQFILQQGLE

[0274] Light chain:AEPMPHSLNFSQYLWYTGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWY QQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTP PTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC

[0275] Purity was >85% as determined by reducing SDS-PAGE. Purity was > 97% as determined by SEC-HPLC. Endotoxin: 0-1 EU / rng. Concentration was 1.29 mg / mL. Formulation buffer was PBS at pH 7.2-7.4.

[0276] As shown in Figure 24, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain and Light chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 31 - Constructing- C20L-Kevzara-L17

[0277] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has Kevzara heavy chain bound to constant heavy (CH) chain domain of Human lgG1 with mutations bound to a linker bound to L17.The light chain sequence has C20 bound to a linker bound to Kevzara light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 38 and the light chain sequence is set out in SEQ ID No. 39, below.

[0278] Heavy Chain:EVQLVESGGGLVQPGRSLRLSCAASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSG RIGYADSVKGRFTISRDNAENSLFLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VTYLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSGGGSMGAQT NFMPMDDLEQRLYEQFILQQGLE

[0279] Light Chain:AEPMPHSLNFSQYLWYTGGGSDIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY QQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFASYYCQQANSF PYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC

[0280] Purity was >85% as determined by reducing SDS-PAGE. Purity was > 68% as determined by SEC-HPLC. Endotoxin: 0-1 EU / mg. Concentration was 1.78 mg / mL. Formulation buffer was PBS at pH 7.2-7.4.

[0281] As shown in Figure 25, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (Heavy chain andLight chain) migrate at about 50-70 kDa and 25-35 kDa, respectively. Non-reduced protein migrates about 150-235 kDa.Example 32 - Testing the anti-cancer efficacy of constructs in a cancer cell line

[0282] The anti-cancer efficacy of constructs was tested in a cancer cell line, MDA-MB-231 using a 24-hour standard MTT assay for cell viability. All constructs were tested at 2.5 pg / mL. The percentage viability of the cancer cells normalized to the negative control for each construct tested is shown in Figure 26. Table 2 shows the statistical analysis comparing the negative control group to each construct. An ANOVA and Dunnett’s multiple comparison test was performed and the mean difference and 95% confidence interval between the viability of construct and control is shown. The threshold is p = 0.05.Table 2. Comparison of viability between constructs and negative control in a cancer cell line.Dunnell's multiple campari eons test Mean Dili. 95.00% Cl of diff. Below th res held? Summary Adjusted P ’Value CFL vj. C20 Prclucnl Yioa 1 ■ 71BJ Ci.riLmt. CR vs. ATF FC4-ENDO (P‘ 25A-L1 / I 241 LEJ ■409311069133 No is I).9 / 62 CFL va. KEVZARA 9933 46009 lu 0436L D.390Li CFt vi CJD-Kevzsm 1514 U 3 Fi / 515 Gf L vs. z20 kEvzara-<ti-L11 66333 iOU l ID iJlUt'U Yes U.U442 CFL vs. I220L Protocol ' / ocul tEGLi 716153 10 50336 No ns ‘O.DOO'J CFL vs. CUOL-Preluent -I t'lSa to 1 ' 4583 No IS 0.2531 CFL v^. AFT TC4-Vdsu ' 6340 J 1330' '.u 540-33 Yes 0.OU3E) CFi vs I ' FH-C,z(H -Kpv / ar.i-kh MM 11 :?[ >384 in 1 (11144 M Yes n nriiih CFL vs. L' / H-Kevzara-kL- Oibl > 334B4 ‘.ti i d8b4y Yes U.UOU I CFL vs. CJO-FCF-VSEO ' 963 KJ -B183 b 12188$ NG IS 0.12G1 C5L vs. |c2U-L1 / JH-Ferceplin Kb q ,7I F to 141783 Yes 0.01U6 CFL vs. L' Fl l-C2'JL-Ht'ueplio i-<i 434. ' to '3 DM 9 Yes 0.94 ' 1 CFt vs D? Hl -HwcBr.hr- 1 1 " ■ tviwr: 415113 / Ti 5 721;.13 Yes 41 114111 CFL vs. MonoNKl (Y 2.^-h-rZ 11 633 r / - I 1 / lU lti I i8344 NO 15 0. 'BUD CFL vs. NK1(Y124A-ATF) UM fi'i ■IU '01 io 170 / 60 Yes '0.UIHJ1 OIL vs. C2OL-Kevzara-L1F 341 KJ -308831099133 No is 0. / 415 CFL vi. IL / 1 ■ I Icrccplln KJ ■10000 9206 / lo 1635)33 Yes '-D.U1J01 CFL vs ATF FC4-Endoip12ial -2799910 T92IJCG No is D t395j CFL va. AFF ■ F C4-£N D O( 3 Iv1} 93' 17 4fiH 04 ‘.u 168 / 49 Yes 0.DO07CFL vs. CJU-Et.vjtont 07 / G7 32.'34 to 162 / 09 Yes 0.9(320

[0283] The following constructs had significantly lower viability of cancer cells compared to the negative control: C20-Kevzara-K5-L17, ATF-Fc4-Vaso1 , L17H-C20L-Kevzara-K5, L17H-Kevzara-K5, (c20-L17H)H-Herceptin-K5, L17H-C20L-Herceptin-K5,C20L-Herceptin-L17, NK1(Y124A-ATF), L17H-Herceptin-K5. ATF-Fc4-Endo(3M) and C20 bivalent.Example 33 - Constructing L17H-C20L-Avastin-L2-P144

[0284] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to L17 bound to a linker bound to Avastin heavy chain bound to constant heavy (CH) chain domain of Human lgG1 bound to a linker bound to P144. The light chain sequence has a signal peptide bound to C20 bound to Avastin light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 40 and the light chain sequence is set out in SEQ ID No. 41 , below.

[0285] Heavy chain:MGWSCIILFLVATATGVHSMGAQTNFMPMDDLEQRLYEQFILQQGLEGGGGEVQLVE SGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAA DFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGST SLDASIIWAMMQN

[0286] Light chain:MGWSCIILFLVATATGVHSAEPMPHSLNFSQYLWYTDIQMTQSPSSLSASVGDRVTITC SASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC

[0287] Purity was >95% as determined by reducing SDS-PAGE. Purity was 97% as determined by SEC-HPLC. Endotoxin: 0.446 Ell / rng. Concentration was 0.13 mg / mL. Formulation buffer was PBS at pH 7.2-7.4.

[0288] As shown in Figure 27, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result different reduced proteins (heavy and light chain) migrate at about 50-75 kDa and 25-37 kDa, respectively. Non-reduced protein migrates about 150-250 kDa.Example 34 - Constructing L17H-C20L-Kevzara-L1-P144

[0289] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to L17 bound to a linker bound to Kevzara heavy chain bound to constant heavy (CH) chain domain of Human lgG1 bound to a linker bound to P144. The light chain sequence has a signal peptide bound to C20 bound to Kevzara light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 42 and the light chain sequence is set out in SEQ ID No. 43, below.

[0290] Heavy chain:MGWSCIILFLVATATGVHSMGAQTNFMPMDDLEQRLYEQFILQQGLEGGGGEVQLVE SGGGLVQPGRSLRLSCAASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYAD SVKGRFTISRDNAENSLFLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSTSLDASIIWAMM QN

[0291] Light chain:MGWSCIILFLVATATGVHSAEPMPHSLNFSQYLWYTDIQMTQSPSSVSASVGDRVTIT CRASQGISSWLAWYQQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQP EDFASYYCQQANSFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

[0292] Purity was >95% as determined by reducing SDS-PAGE. Purity was 96% as determined by SEC-HPLC. Endotoxin: 0 Ell / rng. Concentration was 0.59 mg / mL. Formulation buffer was PBS at pH 7.2-7.4.Example 35 - Constructing L17H-C20L-Kevzara-K5-L1-P144

[0293] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to L17 bound to a linker bound to Kevzara heavy chain bound to constant heavy (CH) chain domain of Human lgG1 bound to a linker bound to K5 bound to a linker bound to P144. The light chain sequence has a signal peptide bound to C20 bound to Kevzara light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 44 and the light chain sequence is set out in SEQ ID No. 43, below.

[0294] Heavy chain:MGWSCIILFLVATATGVHSMGAQTNFMPMDDLEQRLYEQFILQQGLEGGGGEVQLVE SGGGLVQPGRSLRLSCAASRFTFDDYAMHWVRQAPGKGLEWVSGISWNSGRIGYAD SVKGRFTISRDNAENSLFLQMNGLRAEDTALYYCAKGRDSFDIWGQGTMVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSS EEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRN PDGDVGGPWCYTANPRKLYDYCDVPQCAEGGGGSTSLDASIIWAMMQN

[0295] Light chain:MGWSCIILFLVATATGVHSAEPMPHSLNFSQYLWYTDIQMTQSPSSVSASVGDRVTIT CRASQGISSWLAWYQQKPGKAPKLLIYGASSLESGVPSRFSGSGSGTDFTLTISSLQP EDFASYYCQQANSFPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

[0296] Purity was >95% as determined by reducing SDS-PAGE. Purity was 96% as determined by SEC-HPLC. Endotoxin: 0 Ell / rng. Concentration was 0.28 mg / mL. Formulation buffer was PBS at pH 7.2-7.4.Example 36 - Constructing L17H-C20L-Herceptin-K5-L1-P144

[0297] This fusion protein has two heavy chain polypeptides and two light chain polypeptides. The heavy chain sequence has a signal peptide bound to L17 bound to a linker bound to Herceptin heavy chain bound to constant heavy (CH) chain domain of Human lgG1 bound to a linker bound to K5 bound to a linker bound to P144. The light chain sequence has a signal peptide bound to C20 bound to Herceptin light chain bound to a constant human kappa light (CL) chain domain. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified byProtein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 45 and the light chain sequence is set out in SEQ ID No. 46, below.

[0298] Heavy chain:MGWSCIILFLVATATGVHSMGAQTNFMPMDDLEQRLYEQFILQQGLEGGGGEVQLVE SGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGS SEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCR NPDGDVGGPWCYTANPRKLYDYCDVPQCAEGGGGSTSLDASIIWAMMQN

[0299] Light chain:MGWSCIILFLVATATGVHSAEPMPHSLNFSQYLWYTDIQMTQSPSSLSASVGDRVTITC RASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPE DFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC

[0300] Purity was >95% as determined by reducing SDS-PAGE. Purity was 97% as determined by SEC-HPLC. Endotoxin: 0 EU / rng. Concentration was 0.53 mg / mL. Formulation buffer was PBS at pH 7.2-7.4.Example 37- Constructing C20CD16IL15

[0301] This fusion protein has two heavy chain polypeptides. The first heavy chain sequence has a signal peptide bound to C20 bound to a CD16 single variable domain on a heavy chain (VHH) antibody bound to an FC of human lgG1 with a mutation to make a hole and the second heavy chain sequence has a signal peptide bound to C20 boundto IL15 bound to an FC of human lgG1 with a mutation to create a knob. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The first heavy chain sequence is set out in SEQ ID No. 47 and the second heavy chain sequence is set out in SEQ ID No. 48, below.

[0302] Heavy chain 1 : C20-CD16 vhh-Fc lgG1 with hole mutation MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTQVQLQESGGGSVQTGGSLRLSC AASGDTTSEYWGAWFRQAPGKEREAVAAILPLSTTPVYAGSVKGRFTISRDNARNTLY LQMNSLKPEDTAMYYCAAARRGTNAFLTHDKYGYWGQGTQVTVSSEPKSADKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0303] Heavy Chain 2: C20-IL15-FC lgG1 with knob mutationMHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTNWVNVISDLKKIEDLIQSMHIDAT LYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSEPKSADKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0304] Purity was > 95% as determined by reducing SDS-PAGE. Purity was >50% as determined by SEC-HPLC at 280 nm. Endotoxin: 0-1 EU / rng. Concentration was 3.40 mg / mL. As shown in Figure 28, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different reduced proteins (Heavy chain1 and heavy chain 2) migrate at about 40-70 kDa. Non-reduced protein migrates about 70-150 kDa.Example 38- Constructing C20CD16IL15K5

[0305] This fusion protein has two heavy chain polypeptides. The first heavy chain sequence has a signal peptide bound to C20 bound to a CD16 single variable domain on a heavy chain (VHH) antibody bound to an FC of human lgG1 with a mutation to make a hole bound to K5 and the second heavy chain sequence has a signal peptide bound to C20 bound to IL15 bound to an FC of human IgG 1 with a mutation to create a knob bound to K5. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The first heavy chain sequence is set out in SEQ ID No. 49 the second heavy chain sequence is set out in SEQ ID No. 50, below.

[0306] Heavy chain 1: C20-CD16 vhh-Fc lgG1 with hole mutation-K5 MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTQVQLQESGGGSVQTGGSLRLSC AASGDTTSEYWGAWFRQAPGKEREAVAAILPLSTTPVYAGSVKGRFTISRDNARNTLY LQMNSLKPEDTAMYYCAAARRGTNAFLTHDKYGYWGQGTQVTVSSEPKSADKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGG GSGGGGSSEEDCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRA GLEKNYCRNPDGDVGGPWCYTANPRKLYDYCDVPQCAA

[0307] Heavy Chain 2: C20-IL15-FC lgG1 with knob mutation-K5 MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTNWVNVISDLKKIEDLIQSMHIDAT LYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESG CKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSSEE DCMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPETNPRAGLEKNYCRNPD GDVGGPWCYTANPRKLYDYCDVPQCAA

[0308] Purity was > 85% as determined by reducing SDS-PAGE. Purity was >72% as determined by SEC-HPLC at 280 nm. Endotoxin: 0-1 Ell / rng. Concentration was 0.74 mg / mL.

[0309] As shown in Figure 29, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different reduced proteins (Heavy chain and Light chain) migrate as about 50-70 kDa. Non-reduced protein migrates about 100-150 kDa.Example 39- Constructing C20-CD16-1115-Fc1

[0310] This fusion protein has two heavy chain polypeptides. The heavy chain sequence has a signal peptide bound to C20 bound to a CD16 single variable domain on a heavy chain (VHH) antibody bound to a linker bound to IL15 bound to an Fc of Human lgG1 with C220A. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 51, below.MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTQVQLQESGGGSVQTGGSLRLSC AASGDTTSEYWGAWFRQAPGKEREAVAAILPLSTTPVYAGSVKGRFTISRDNARNTLY LQMNSLKPEDTAMYYCAAARRGTNAFLTHDKYGYWGQGTQVTVSSPSGQAGAAASE SLFVSNHAYNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TSEASGGPEEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK

[0311] Purity was > 95% as determined by reducing SDS-PAGE. Purity was >75% as determined by SEC-HPLC at 280 nm. Endotoxin: 0-1 Ell / rng. Concentration was 0.72 mg / mL.

[0312] As shown in Figure 30, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result the reduced protein migrates at about 70-100 kDa. Non-reduced protein migrates about 150-235 kDa.Example 40- C20-CD16-1115-Fc1-K5

[0313] This fusion protein has two heavy chain polypeptides. The heavy chain sequence has C20 bound to a CD16 single variable domain on a heavy chain (VHH) antibody bound to a linker bound to IL15 bound to an Fc of Human lgG1 with C220A bound to K5. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The heavy chain sequence is set out in SEQ ID No. 52, below.MHSSALLCCLVLLTGVRAAEPMPHSLNFSQYLWYTQVQLQESGGGSVQTGGSLRLSC AASGDTTSEYWGAWFRQAPGKEREAVAAILPLSTTPVYAGSVKGRFTISRDNARNTLY LQMNSLKPEDTAMYYCAAARRGTNAFLTHDKYGYWGQGTQVTVSSPSGQAGAAASE SLFVSNHAYNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSEASGGPEEPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVW DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGGSGGGGSSEEDCMFGNGKGYRGKRATTVTGTPCQD WAAQEPHRHSIFTPETNPRAGLEKNYCRNPDGDVGGPWCYTANPRKLYDYCDVPQC AA

[0314] Purity was > 85% as determined by reducing SDS-PAGE. Purity was >89% as determined by SEC-HPLC at 280 nm. Endotoxin: 0-1 Ell / rng. Concentration was 0.29 mg / mL.

[0315] As shown in Figure 31, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 2) and non-reduced (Lane 3) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result the reduced protein migrates at about 70-100 kDa. Non-reduced protein migrates about 150-235 kDa.Example 41- ATF-Arrestin-FC (lgG4)

[0316] This fusion protein has a signal peptide bound to ATF bound to a linker bound to arresten bound to a human lgG4 Fc. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The sequence is set out in SEQ ID No. 53, below.MEWSWVFLFFLSVTTGVHSSNELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKF GGQHCEIDKSKTCYEGNGHFYRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQ LGLGKHNYCRNPDNRRRPWCYVQVGLKPLVQECMVHDCADGSGSGSSVDHGFLVT RHSQTIDDPQCPSGTKILYHGYSLLYVQGNERAHGQDLGTAGSCLRKFSTMPFLFCNI NNVCNFASRNDYSYWLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAMVMAVHSQTI QIPPCPSGWSSLWIGYSFVMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTC NYYANAYSFWLATIERSEMFKKPTPSTLKAGELRTHVSRCQVCMRRTPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQV YTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0317] Purity was > 90% as determined by reducing SDS-PAGE. Endotoxin: <1 Ell / mg. Concentration was 1.5 mg / mL. The formulation buffer was PBS, pH 7.4.

[0318] As shown in Figure 32, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result, the reduced protein migrates at about 63-75 kDa.Example 42- ATF-hulgG4 Fc-Arrestin

[0319] This fusion protein has a signal peptide bound to ATF bound to a linker bound to human lgG4 Fc bound to a linker bound to arresten. The molecule was produced using standard molecular biology techniques and expressed using mammalian cell culture. The expression vectors of the fusion protein were transiently transfected and expressed in mammalian cells with chemically defined culture media. This protein was purified by Protein A affinity chromatography, dialysed with PBS, concentrated (if needed), and then subjected to 0.2 micron sterile filtration to get the bulk with high purity. The sequence is set out in SEQ ID No. 54, below.NELHQVPSNCDCLNGGTCVSNKYFSNIHWCNCPKKFGGQHCEIDKSKTCYEGNGHF YRGKASTDTMGRPCLPWNSATVLQQTYHAHRSDALQLGLGKHNYCRNPDNRRRPW CYVQVGLKPLVQECMVHDCADGSGSGSPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLH QDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSSVDHGFLVTRHSQTIDDPQ CPSGTKILYHGYSLLYVQGNERAHGQDLGTAGSCLRKFSTMPFLFCNINNVCNFASRN DYSYWLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAMVMAVHSQTIQIPPCPSGWSSLWIGYSFVMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNYYANAYSFW LATIERSEMFKKPTPSTLKAGELRTHVSRCQVCMRRT

[0320] Purity was > 95% as determined by reducing by SEC-HPLC. Endotoxin: <1 Ell / mg. Concentration was 2 mg / mL. Formulation buffer PBS, pH 7.4.

[0321] As shown in Figure 33, SDS-PAGE analysis of the construct in [3-mercaptoethanol-reduced (Lane 1) and non-reduced (Lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result, the reduced protein migrates at about OSS kDa. The non-reduced protein migrates about 180 kDa.

Claims

1. WHAT IS CLAIMED IS:

1. A compound comprising:- A first binding peptide having anti-angiogenic or anti-cancer activity operatively linked to an N-terminus of a heavy chain and / or a light chain polypeptide, wherein- the heavy chain polypeptide comprises a heavy chain region of an antibody used to treat cancer or ocular angiogenesis operatively linked to a first immunoglobulin (IgG) antibody peptide and- the light chain polypeptide comprises a light chain region of an antibody used to treat cancer or ocular angiogenesis operatively linked to a second immunoglobulin (IgG) antibody peptide.

2. The compound of claim 1 comprising two heavy chain polypeptides and two light chain polypeptides.

3. The compound of claim 1 comprising two heavy chain polypeptides and one light chain polypeptide.

4. The compound of the preceding claims, wherein the second IgG antibody peptide is a human kappa constant light (CL) chain.

5. The compound of claim 1 comprising two heavy chain polypeptides.

6. The compound of the preceding claims, wherein the first IgG antibody peptide is a fragment of an Fc fusion peptide.

7. The compound of the preceding claims, wherein the first IgG antibody peptide is a fragment of a human Fc fusion peptide.

8. The compound of claims 6-7, wherein the Fc fusion peptide is an IgG protein.

9. The compound of claims 6-8, wherein the Fc fusion peptide is selected from the group consisting of an lgG1 isotype Fc peptide, an lgG2 isotype Fc peptide, an lgG3 isotype Fc peptide, an lgG4 isotype Fc peptide.

10. The compound of claims 1-5, wherein the first IgG antibody peptide is a constant heavy chain peptide of human lgG1.

11. The compound of claim 10, wherein the first IgG antibody peptide is a constant heavy chain peptide of human lgG1 with one or more amino acid mutations.

12. The compound of the preceding claims wherein the first binding peptide comprises C20.

13. The compound of claims 1-11 wherein the first binding peptide comprises one of L17H, P144 or G3-A3.

14. The compound of the preceding claims further comprising a second binding peptide having anti-angiogenic or anti-cancer activity operatively linked to the N- terminus or C-terminus of the heavy chain and / or the light chain polypeptide.

15. The compound of claim 14, wherein the second binding peptide comprises one of C20, L17H, P144 or G3-A3.

16. The compound of the preceding claims, wherein the heavy chain region of an antibody used to treat cancer or ocular angiogenesis comprises one of Praluent heavy chain, Kevzara heavy chain, Herceptin heavy chain or Avastin heavy chain.

17. The compound of the preceding claims, wherein the light chain region of an antibody used to treat cancer or ocular angiogenesis comprises one of Praluent light chain, Kevzara light chain, Herceptin light chain or Avastin light chain.

18. The compound of the preceding claims further comprising, a third binding peptide comprising an endostatin derived peptide sequence having endostatin activity or a plasminogen derived peptide sequence having plasminogen activity or a hepatocyte growth factor derived peptide having hepatocyte growth factor antagonist activity or a vasostatin derived peptide having vasostatin activity, operatively linked to the C-terminus of the heavy chain polypeptide.

19. The compound of claim 18, wherein the third binding peptide is selected from a group consisting of a fragment of an endostatin having endostatin activity, an endostatin, a fragment of a plasminogen having plasminogen activity, an endostatin with a P125 mutation, a kringle domain of plasminogen, kringle domains 1 to 5 of plasminogen, a kringle domain 5 (K5) from a plasminogen, an NK1 domain of hepatocyte growth factor with a Y124A mutation or a vasostatin 1.

20. A compound comprising two heavy chain polypeptides coded by a heavy chain sequence and two light chain polypeptides coded by a light chain sequence selected from a group of heavy chain sequence and light chain sequence combinations comprising SEQ ID No. 1 and SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4, SEQ ID No. 1 and SEQ ID. No. 4, SEQ ID No. 5 and SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, SEQ ID No. 12 and SEQ ID No. 10, SEQ ID No. 13 and SEQ ID No. 10, SEQ ID No. 14 and SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No. 15, SEQ ID No. 17 and SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20, SEQ ID No. 21 and SEQ ID No. 8, SEQ ID No. 22 and SEQ ID No. 10, SEQ ID No. 25 and SEQ ID No. 10, SEQ ID No. 26 and SEQ ID No. 10, SEQ ID No. 27 and SEQ ID No. 10, SEQ ID No. 28 and SEQ ID No. 10, SEQ ID No. 29 and SEQ ID No. 30, SEQ ID No. 9 and SEQ ID No. 30, SEQ ID No. 33 and SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 36, SEQ ID No. 37 and SEQ ID No. 34, SEQ ID No. 38 and SEQ ID No. 39, SEQ ID No. 40 and SEQ ID No. 41, SEQ ID No. 42 and SEQ ID No. 43, SEQ ID No. 44 and SEQ ID No. 43, or SEQ ID No. 45 and SEQ ID No. 46.

21. A compound comprising a compound at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the compound of claim 20.

22. A compound selected from the group consisting of C20H-Herceptin-K5, C20L- Herceptin-K5, C20HL-Herceptin-K5, Tetravalent C20-Fc4, C20H-Kevzara-K5, Praluent-Endo(P125A), C20-Praluent-Endo(P125A), C20-Praluent, L17H-C20L- Avastin-Endo, L17H-C20L-Avastin-K5, L17H-C20L-Kevzara-K5, C20L-Kevzara- L17-Endostatin(P125A), C20H-Kevzara-HGF(Y124Y)NK1 , C20-Praulent- Vasostatinl, C20-Kevzara, C20-Kevzara-K5-L17, C20-Kevzara-Vasostatin1-L17, C20-Kevzara-Endostatin(p125A)-L17, C20L-Praluent-Vasostatin1 , C20L- Praluent, L17H-C20L-Herceptin-K5, (C20-L17)H-Herceptin-K5, C20L-Herceptin- L17, C20L-Kevzara-L17, L17H-C20L-Avastin-L2-P144, L17H-C20L-Kevzara-L1- P144, L17H-C20L-Kevzara-K5-L1-P144 and L17H-C20L-Herceptin-K5-L1-P144.

23. A compound comprising Praluent-Endo(P125A).

24. A compound having two heavy chain polypeptides and two light chain polypeptides, wherein the heavy chain polypeptides are coded by a heavy chain sequence comprising SEQ ID. NO. 11 and the light chain polypeptides are coded by a light chain sequence comprising SEQ ID. No. 10.

25. A compound selected from the group consisting of ATF-Fc4-Endostatin(P125A)- L17H, ATF-Fc4-Vasostatin1, ATF-Arrestin-FC (lgG4) and ATF-hulgG4 Fc- Arrestin.

26. A compound comprising C20 Bivalent.

27. A compound selected from the group consisting of C20CD16IL15, C20CD16IL15K5, C20-CD16-1115-Fc1 and C20-CD16-1115-Fc1-K5.

28. A compound having two heavy chain polypeptides, wherein the heavy chain polypeptides are coded by a heavy chain sequence selected from SEQ ID No. 23, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 51, SEQ ID No. 52, SEQ ID No.53 or SEQ ID No. 54.

29. A compound comprising a first heavy chain polypeptide coded by a first heavy chain sequence and a second heavy chain polypeptide coded by a second heavy chain sequence selected from a group of first heavy chain sequence and second heavy chain sequence combinations comprising SEQ ID No. 47 and SEQ ID No.48 or SEQ ID No. 49 and SEQ ID No. 50.

30. A compound comprising a compound substantially homologous to the compound of the preceding claims and having the activity of the compound of the preceding claims.

31. One or more nucleotides encoding the compound of the preceding claims.

32. A vector containing the compound of the preceding claims.

33. The compound of the preceding claims, wherein the compound exhibits anticancer activity.

34. The compound of the preceding claims, wherein the compound exhibits anti- angiogenic activity to treat eye diseases caused by ocular angiogenesis.

35. A method of treating cancer comprising administration of a composition comprising the compound of the preceding claims.

36. A use of the compound of the preceding claims for treatment of cancer.

37. A method for inhibiting metastasis in a subject with cancer, the method comprising administering an effective amount of the compound of the preceding claims to the subject.

38. The method or use of the preceding claims, further comprising one or more of the following a) administering chemotherapy; b) administering radiation therapy; or c) administering one or more additional therapeutic agents.

39. The method or use of claims 35-38, wherein the cancer is a solid cancer.

40. A method of treating eye diseases caused by ocular angiogenesis comprising administration of a composition comprising the compound of the preceding claims.

41. A use of the compound of the preceding claims for treatment of eye diseases caused by ocular angiogenesis.

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